http://2013.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=AlexBates&year=&month=2013.igem.org - User contributions [en]2024-03-28T22:24:53ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UCL/Practice/CreativeTeam:UCL/Practice/Creative2013-11-06T18:33:18Z<p>AlexBates: </p>
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<p class="major_title">Creative Writing Competition: 'Changing the Human Brain'</p><br />
</p><br />
<p class="minor_title">Opening Ethical Windows Into The Human Mind</p><br />
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<p class="body_text"><br />
Science fiction is, perhaps, the greatest liar the universe has ever known. Where are our <a href="http://www.imdb.com/title/tt1219289/" target="_blank">'limitless'</a> pills that super excite our brains into genius? Where is the <a href="http://www.imdb.com/title/tt0338013/" target="_blank"> eternal sunshine</a> of the memory eraser machine and where is the fiery ‘nervous system upgrade’ technology featured in Iron Man 3? Ever since <a href="http://www.wired.com/wiredscience/2012/03/what-can-novelists-learn-from-neuroscience" target="_blank">science met fiction</a> writers have envisioned possible, if not always plausible, technologies to come and this has fed right back into science, inspiring generations of new researchers and moulding the public’s perception of what goes on under the fume hood and in the petri dish. Sometimes it gets its predictions right, as with the touchscreen of Star Trek or the point-of-view guns of Douglas Adam’s, but right or wrong it plays a key role in demonstrating public opinion and controlling it.<br />
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<p class="body_text"><br />
Fiction gives us an <a href="http://pieceslight.blogspot.co.uk/2013/05/neuroscience-in-fiction.html" target="_blank">unparalleled medium</a> through which to comprehend the value of neuroscientific accounts of behaviour and experience, because it allows for a very human non-scientific study of the effects of neuroscience, from the point of view of the very minds encountering new fandangled technologies. If genetic engineering of the brain really does perturb our sense of selfhood, help us fight mental diseases or endow us with new abilities, writers will swarm to produce work that can act as an ethical window into these nascent technologies. Their fiction can tell us something about how we consume, as a society, scientific ideas and blend them with social philosophy.<br />
</p><br />
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<br />
<p class="body_text"><br />
This is the thinking, along with the animated discussions at our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">speed debate</a>, that inspired us to run a creative writing competition on the topic ‘changing the human brain/mind’. The competition ran from the 14th of August to September the 15th, and we received over fifty entries. Writers were allowed to submit short stories of 500-1,500 words, poems of up to 40 lines and (screen)plays of up to a 30 minute run time. The winning entries, along with a commentary by a UCL scientist, can be found below, in alphabetical order.<br />
</p><br />
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<p class="body_text"><br />
<b>The UCL iGEM 2013 Spotless Mind team would like to thank everyone who has sent their entries to us. It's a wonderful thing to receive submissions from many countries all around the world, knowing that our project has reached different corners of the globe! We are humbled by your interest in our project, and we hope these winning entries will serve as inspiration to people passionate in both literature and science.</b><br />
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<p class="major_title">Winning Entries</p><br />
</p><br />
<p class="minor_title">Congratulations!</p><br />
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<p class="body_text"><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/5/5b/Natasha_Ali.pdf" target="_blank">Anamnesis by Natasha Ali</a><br />
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</p><br />
<p class="body_text"><br />
<I>Swallow, says the hawk. She gestures at the plate in front of me. It is grey, but then the plates are always grey. Our clothes are always white and starched and uncomfortable. Our stools are always cold when we sit on them, our feet grazing the colder floor...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: Natasha Ali was born in Karachi, Pakistan, and raised both there and in Brussels, Belgium, but currently lives in Riyadh, KSA. She is about to start her last year at a sixth-form college. The trials and tribulations of scientific research have always fascinated her.</b><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/a/a5/Paul_Aroniyo.pdf" target="_blank">Moving Too Fast by Paul Aroniyoi</a></p><br />
</p><br />
<p class="body_text"><br />
<I>Doctor says the effects of the drugs will soon fade, as my brain reconfigures itself and adjusts to accommodating; I quote “Higher levels of cognitive thinking.” But I’m sure weeks have passed and yet these headaches and the nausea still persist; I’m not getting any better and I think the Doc knows it...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: I’m currently a Creative Writing student and I’m from London. I’m an avid sci-fi and comic book enthusiast (Yes, which means I love that characters but don’t read the actual comics!).</b><br />
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<div class="gap"></div><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/a/af/Dot_Cobely.pdf" target="_blank">Four Poems by Dot Cobley</a></p><br />
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<p class="body_text"><br />
<I>There’s an intriguing illustration<br />
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<p class="body_text"><br />
on this leaflet that the neuro team gave us.<br />
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<p class="body_text"><br />
Looks like somebody had fun<br />
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<p class="body_text"><br />
playing around with those little plastic wheels...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: Dot Cobley’s poems appear in numerous anthologies and magazines, including The Rialto, Smiths Knoll and The SHOp. She underwent neurosurgery for trigeminal neuralgia, and has recently been diagnosed with Parkinson’s.</b><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/4/4d/Siân_Davies.pdf" target="_blank">Affordable Beauty by Siân Davies</a></p><br />
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<p class="body_text"><br />
<I>There was electric classical music playing in the elevator when Alan Winterm alighted, his eyes fixed on the pre-released PDA in his hand. The silver ring he wore glinted in the cold, artificial light, dancing in his peripheral vision as he sent emails, replied to messages, booked appointments...</I><br />
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<p class="body_text"><br />
<b>Writer's biography: My name is Siân, it's Welsh but I'm not. I live in a tiny rural town in Shropshire, selling people kettles and trying to learn how to write.</b><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/d/da/Carol_Fraser.pdf" target="_blank">A Change of Mind by Carol Fraser</a></p><br />
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<p class="body_text"><br />
<I>Just suppose. Now let's imagine. What if?<br />
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<p class="body_text"><br />
What would it be like to have a memory<br />
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<p class="body_text"><br />
That functioned sometimes but at others failed the test, <br />
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Like some old creaky household gadget on the blink, <br />
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As trusty as a teacup made of lace?<br />
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<p class="body_text"><br />
...</I><br />
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<p class="body_text"><br />
<b>Writer's biography: I am a retired musician, now a (very) mature philosophy student. I am interested in every aspect of human condition - and that of non-human.</b><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/c/c4/Hilary_Greenleaf.pdf" target="_blank">284 steps by Hilary Greenleaf</a></p><br />
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<p class="body_text"><br />
<I>The past has become an area of conflict, a dangerous area of uncertainty that lies extinct yet threatening, waiting to draw us all into fresh conflict and pain. As a family we are learning to sidestep it, and something that should be so natural for people with a shared history is now taboo...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: Hilary Greenleaf (46) is an HCPC registered podiatrist and mother of two. She lives in the Essex countryside and writes short stories in her spare time.</b><br />
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<br />
<div class="gap"></div><br />
<br />
<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/8/86/Fatima_Muhammad.pdf" target="_blank"> The Demolishing Change by Fatima Muhammad</a></p><br />
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<p class="body_text"><br />
<I>They looked trivial. He knew the crowd was made up of individuals, each one with a story, each life holding value, but what of it? Together they made up an apathic crowd. One which, from his perspective, looked trivial...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: Fatima Muhammad is a doctor, currently doing a postgraduate degree in Medical Education from Cardiff University. She’s had a few short stories published. Nothing large-scale yet, but here’s hoping.</b><br />
<br />
<div class="gap"></div><br />
<br />
<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/2/2f/Martha_Patterson.pdf" target="_blank"> A Constant Man by Martha Patterson</a></p><br />
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</p><br />
<p class="body_text"><br />
<I>As he ages, Johnny comes to grips with his mother's dementia and her anxieties about her marriage...</I><br />
<br />
</p><br />
<p class="body_text"><br />
<b>Writer's biography: Martha Patterson has written more than 100 plays and has had work published in four anthologies by the International Centre for Women Playwrights and several collections by JAC Publishing and Original Works Publishing. Her work has been produced Off-Off-Broadway and in the UK, Korea, and Australia, as well as in twelve states around the USA. She has also had a half-hour mystery produced by Shoestring Radio Theatre in San Francisco. She earned her B.A. from Mount Holyoke College and an M.A. from Emerson College, both degrees in Theatre. She is a member of the Dramatists Guild of America, the International Centre for Women Playwrights, Screen Actors Guild, and Actors’ Equity Association. She lives in Boston, Massachusetts.</b><br />
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<p class="body_text"><b> <a href="https://static.igem.org/mediawiki/2013/4/4e/Ng_Chin_San.pdf" target="_blank">Changing the Human Brain by Ng Chin San</a></p><br />
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<p class="body_text"><br />
<I>The road below was scattered with litter, broken beer bottles and picket signs the only evidence left of the chaos that was the previous evening. The Scientist stood motionless at the window surveying the dark scene, taking a sip from a glass of whiskey in his hand from time to time as Beethoven’s piano sonata in G minor played softly in the background...</I><br />
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</p><br />
<p class="body_text"><br />
<b>Writer's biography: Ng Chin San is a third year law student at UCL. He enjoys playing sports. He also enjoys sleeping.</b><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:58:34Z<p>AlexBates: </p>
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<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
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<p class="major_title">Bacterial Lab Protocols</p><br />
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<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
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<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
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</table><br />
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<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
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<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
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<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
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<td>RO HCL</td><br />
<td>500ml</td><br />
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<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
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<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
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<td>1M CaCl2</td><br />
<td>5</td><br />
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<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
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<td>RO H2O</td><br />
<td>37.5</td><br />
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<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
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<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
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<td>5x M9 Salts</td><br />
<td>10mL</td><br />
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<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
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<td>20% D Glucose</td><br />
<td>1 mL</td><br />
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<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
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<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
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<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
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<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
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</table><br />
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<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
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<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
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<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
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<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
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<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
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<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
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<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
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<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>10)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7e/Labook_lab_2013.jpg');height:369px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d6/Tubes_lab_2013.jpg');height:360px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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<div class="full_page"><br />
<p class="major_title">IGEM: INTELLIGENTLY GENETICALLY ENGINEERED MICROGLIA</p><br />
<p class="minor_title">Synthetic Biology Fights Alzheimer's Disease</p><br />
<br />
<p class="abstract_text" style="color:#404040;"><br />
This year, the UCL iGEM team is taking a radical new step with synthetic biology. We intend to explore the potential application genetic engineering techniques on the brain, because it is the site of some of the most subtle, and many of the most devastating medical conditions. Alzheimer’s Disease is a neurodegenerative disease characterised by the loss of recent memory and intellectual functions. We have devised a genetic circuit for transfection into microglia, a novel chassis in which standard assembly has never been used, to boost their ability to break down senile plaques, which are associated with Alzheimer’s disease, as well as to support and protect endangered neurons from microglia-mediated neuroinflammation.<br />
<br><br><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCLTeam:UCL2013-10-05T03:56:19Z<p>AlexBates: Undo revision 294439 by Kckiew (talk)</p>
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<div class="full_page"><br />
<p class="major_title">IGEM: INTELLIGENTLY GENETICALLY ENGINEERED MICROGLIA</p><br />
<p class="minor_title">Synthetic Biology Fights Alzheimer's Disease</p><br />
<br />
<p class="abstract_text" style="color:#404040;"><br />
<br />
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<div class="gap"><br />
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7e/Labook_lab_2013.jpg');height:369px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d6/Tubes_lab_2013.jpg');height:360px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:52:40Z<p>AlexBates: </p>
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7e/Labook_lab_2013.jpg');height:369px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d6/Tubes_lab_2013.jpg');height:360px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:52:00Z<p>AlexBates: </p>
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
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<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
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<div class="gap"><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
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<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
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<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
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<div class="gap"></div><br />
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<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7e/Labook_lab_2013.jpg');height:369px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d6/Tubes_lab_2013.jpg');height:360px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/File:Tubes_lab_2013.jpgFile:Tubes lab 2013.jpg2013-10-05T03:50:46Z<p>AlexBates: </p>
<hr />
<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:48:03Z<p>AlexBates: </p>
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7e/Labook_lab_2013.jpg');height:369px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/File:Labook_lab_2013.jpgFile:Labook lab 2013.jpg2013-10-05T03:47:23Z<p>AlexBates: </p>
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<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:44:27Z<p>AlexBates: </p>
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"><div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Labbook/Week16Team:UCL/Labbook/Week162013-10-05T03:41:54Z<p>AlexBates: </p>
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<br />
<p class="major_title">Lab Weeks</p><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_page"><br />
<p class="body_text"> <a href="https://2013.igem.org/Team:UCL/LabBook/Week1">Week 1</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week2"> Week 2</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week3"> Week 3</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week4"> Week 4</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week5"> Week 5</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week6"> Week 6</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week7"> Week 7</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week8"> Week 8</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week9"> Week 9</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week10"> Week 10</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week11"> Week 11</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week12"> Week 12</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week13"> Week 13</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week14"> Week 14</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week15"> Week 15</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week16"> Week 16</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week17"> Week 17</a> | <a href="https://2013.igem.org/Team:UCL/Labbook/Week18"> Week 18</a> <br />
</p> <br />
</div><br />
<br />
<p class="minor_title">Week 16</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Bacterial Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Monday 16th September</b><br />
</p><br />
<p class="body_text"><br />
Results of the inoculations of transformations of 13/09 in 4xcmp: all showed growth apart from falcons 5.3, 5.2, 1.14 and 1.2. Glycerol stocks of the rest of 19 inoculations were made.<br />
</p><br />
<p class="body_text"><br />
Minipreps of the above inoculations were made only for 1.3, 1.7, 1.9, 1.10, 1.11, 1.15, 1.20 and 5.1 due to lack of chromatographic columns.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of the above minipreps showed concentration values below 16.0 ng/ul.<br />
Analytical digest of miniprep samples 1-9 (prepared the day before) as well as of 5.1 and 1.15 (which showed concentrations of about 15 ng/ul) with E and P<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)<br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>1.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/9/9c/Andymon_was_here.png');height:515px;width:650px"></div><br />
</p><br />
<p class="body_text"><br />
Prep digest of miniprep of J63009 with E & P in order to keep up the pSB1C3 stocks<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Cut</th><br />
<th>Uncut</th><br />
<th>Control - pSecTag2A</th><br />
</tr><br />
<tr><br />
<td>J63009DNA</td><br />
<td>25</td><br />
<td>5</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>EocR1</td><br />
<td>2</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>2</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>4</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>6.5</td><br />
<td>5</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>40</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated for 2 hours at 37O C.<br />
Casey ran a gel of the following samples in the next order:<br />
Zeo BB: 26D, 26u; 28D, 28u; AuxD, AuxU <br />
</p><br />
<p class="body_text"><br />
Gel1: E+P double digest and P, single digest (Auxin bb): 3, 12, 13, 15.<br />
Gel2: D digest 26, 28 and Auxin bb.<br />
Minipreps: 26, 28, 3, 12, 13, 15, 21 (sample of which DNA is waiting to be eluted)<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 17th September</b><br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols"> Miniprep</a> of the 16 tubes of inoculations of MMP9 as well as tube 21 of which DNA had to be eluted.<br />
</p><br />
<p class="body_text"><br />
Nanodrop results of the minipreps<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube no.</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>20.6</td><br />
<td>1.9</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>26.9</td><br />
<td>1.64</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>22.0</td><br />
<td>3.2</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>4.8</td><br />
<td>2.16</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>22.7</td><br />
<td>2.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>16.4</td><br />
<td>2.41</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>16.9</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>7.1</td><br />
<td>1.49</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>27.3</td><br />
<td>1.79</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>16.0</td><br />
<td>1.72</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>22.2</td><br />
<td>1.41</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>11.0</td><br />
<td>1.73</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>12.0</td><br />
<td>1.6</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>18.4</td><br />
<td>1.57</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>45.1</td><br />
<td>1.62</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>9.2</td><br />
<td>1.72</td><br />
</tr><br />
<tr><br />
<td>21 (kc)</td><br />
<td>21.3</td><br />
<td>1.81</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Nanodrop readings (Tom's)<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Sample</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>J63A (glyc stock)</td><br />
<td>62.3</td><br />
<td>1.74</td><br />
</tr><br />
<tr><br />
<td>CCB4 (4xcmp comp cells)</td><br />
<td>46.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>CCB2 (2xcmp comp cells)</td><br />
<td>57.6</td><br />
<td>1.87</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Analytical digest of zeo+pSB1C3 (from AA1 ligation) potential clones with xba1<br />
Eight clones (AA1 col x miniprep 15/09 RC) + AA1 5xcmp mini prep were digested with xba1 following the recipe:<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Xba1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Samples were briefly centrifuged and incubated at 37O C for circa 3 hours. After that, samples were supplemented with 3 ul dye and run on a gel.<br />
</p><br />
<p class="body_text"><br />
<u>Purification of 8 PCR reactions to amplify Zeo and BB hangers</u><br />
</p><br />
<p class="body_text"><br />
These reactions (total volume of 400 ul) were left in the thermocycler at 4O C overnight. The samples were run on a gel together with 70 ul dye and the correct bands (1.8 kb) were gel extracted. This gel was purified and eluted in 40 ul Elution Buffer. The nanodrop readings of these were of 91.9 ul/ul with purity (260/280) of 1.96.<br />
</p><br />
<p class="body_text"><br />
<u>Preparative digest of amplified zeocin using EcoR1 and Pst1</u><br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volume (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>35</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>37</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These reactions were Incubated for 2 hours at 37OC.<br />
After, the digest was purified with PCR purification kit. Nanodrop result was found to be 60.4 ng/ul (260/280=1.82, the purity).This was taken further for a ligation as per the following recipe:<br />
</p><br />
<p class="body_text"><br />
<u>Ligation 5 for zeocin and pSB1C3</u><br />
</p><br />
<p class="body_text"><br />
pSB1C3 concentration = 50 ng/ul<br />
</p><br />
<p class="body_text"><br />
Zeocin insert concentration = 25 ng/ul<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Lig 1 (ul)</th><br />
<th>Lig 2 (ul)</th><br />
<th>Lig 3 (ul) control</th><br />
<th>Lig 4 (ul) control</th><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Zeo</td><br />
<td>2</td><br />
<td>2.5</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>Quick T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
<td>4.5</td><br />
<td>7</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>20</td><br />
<td>20</td><br />
<td>20</td><br />
<td>20</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These tubes were incubated at room temperature for 5 minutes then used 5 ul from each for transformation using W3110 cells. Plated 10 ul, 90 ul on 5xcmp selective plates and then incubated overnight at 37°C. Next day, there was no growth on neither plates<br />
</p><br />
<p class="body_text"><br />
Two gel were loaded using xba1 digests of recombinant zeo candidates<br />
</p><br />
<p class="body_text"><br />
Ten ul of sample 1(showed correct band pattern) was used from glycerol stock to make an inoculation in 4xcmp 10 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<u>Inoculation of MMP9 glycerol stocks</u><br />
</p><br />
<p class="body_text"><br />
These were made using 2 ml LB broth, 8 ul material from the glycerol stock and 4xcmp (8 ul cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 18th September</b><br />
</p><br />
<p class="body_text"><br />
Carried out <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> miniprep</a><br />
of 16 MMP9 glycerol stocks candidates which were inoculated overnight the day before; another miniprep was set for 2 samples from zeocin ligation 1 (prepared the day before) which was inoculated overnight (10 ml LB and 40 ul cmp).<br />
Nanodrop results of the above<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube label</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>97.6</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>151.8</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>121.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>103.6/td><br />
<td>1.83</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>104.8</td><br />
<td>1.92/td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>409.2</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>220.3</td><br />
<td>1.79</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>187.3</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>188.2</td><br />
<td>1.91</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>82.8/td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>79.5</td><br />
<td>1.76</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>170.6/td><br />
<td>1.73</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>117.1</td><br />
<td>1.92</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>177.8</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>119.7</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>167.5</td><br />
<td>1.69</td><br />
</tr><br />
<tr><br />
<td>Zeo lig1A</td><br />
<td>419.6</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>Zeo lig1B</td><br />
<td>97.2</td><br />
<td>2.03</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Casey single digests recipes<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Component</th><br />
<th>Xba1 digest (ul)</th><br />
<th>EcoR1 digest (ul)</th><br />
<th>Pst1 digest (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Xba1</td><br />
<td>1</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>-</td><br />
<td>1</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>-</td><br />
<td>-</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4/3</td><br />
<td>1 (buffer 5)</td><br />
<td>1 (EcoR1 buffer)</td><br />
<td>1 (buffer 3)</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
</total><br />
</p><br />
<br />
<p class="body_text"><br />
Gel loading order wells: 1x, 1E, 1P, 1u, 13x, 13E, 13P, 13u, 21x, 21E, 21P, 21u, 1.15x, 1.15E, 1.15P, 1.15u, 5.1x, 5.1E, 5.1P, 5.1u.<br />
</p><br />
<p class="body_text"><br />
Carried out gel extraction and purification of Weiling’s MMP9 amplification; total material loaded: 2x50 ul vials from past PCR and 6x50 ul PCR left at 4oC overnight on 17/09/13.<br />
After the purification procedure, the concentration of MMP9 was found to be 111 ng/ul and 260/280 indices = 2.13.<br />
</p><br />
<p class="body_text"><br />
<u>Prep digest of purified MMP9 with Dpn1, EcoR1 and Pst1</u><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>58</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Ecor1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Dpn1</td><br />
<td>7</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated for 2 hours at 37C. This was followed be a PCR purification. <br />
</p><br />
<p class="body_text"><br />
<u>Prep digest of pSB1C3 (the entire stock) with Dpn1, EcoR1, Pst1</u><br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>70</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Ecor1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Dpn1</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>100</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
The digestion was incubated for 2 hours and then incubated for 20 min.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 19th September</b><br />
</p><br />
<p class="body_text"><br />
Focus on MMP-9<br />
- Minipreped samples from the 18 inoculations, only 16 of these had growth.<br />
- Took the 16 inoculations forward to minipreping.<br />
- Made glycerol stocks of the 16 inoculations.<br />
- Took nanodrop readings (range varied between 6 ng/ul -45 ng/ul).<br />
- Did an analytical digest with Xba1 using 5 samples of the highest concentrations (2, 4, 10, 14, 17).<br />
- Gel result didn’t show bands for 4, 2, 14, 17, only sample 10 showed visible bands for both cut and uncut.<br />
- Re-inoculated 16 samples from glycerol stocks for 16 hours to for minipreps the following day.<br />
- re-PCRed 6 tubes of MMP-9 with MMP9 4 bb RseFW primes (total of 8 tubes to gel extract and purify the following day).<br />
Nanodrop of PCR purified and E+P+D digest PSBIC3 (2013 High school iGEM )and MMP9 insert<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9</td><br />
<td>184.4</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>27.4</td><br />
<td>1.73</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Xba1 restriction sites in PSBIC3 – 1 restriction site<br />
</p><br />
<p class="body_text"><br />
the recombinant plasmid (with zeocin as an insert) is about 3-6-3.8 kb when cut with Xba1 or EcoR1.<br />
Instead the gel run for the xba1 digests have a strong 2 kb band in all the cuts. Possibly, some plasmids may have ligated to themselves.<br />
</p><br />
<p class="body_text"><br />
<u>Repeat digest of minipreps of AA1 ligations (transformation of only using EcoR1)</u><br />
</p><br />
<p class="body_text"><br />
Samples digested (9 in total): AA1 miniprep col 1à 8 & AA1 5xcmp miniprep<br />
10µl reaction volume<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Components</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>EcoR1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
</tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>2.5</td><br />
</tr><br />
<td>Total</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/0/04/Weilingyuan.png');height:315px;width:666px"></div><br />
<p class="body_text"><br />
Re-Inocubations from glycerol stocks from both batches of <a href="https://2013.igem.org/Team:UCL/Project/Protocols"> transformation</a> with AA1 ligation pick using:<br />
- 2ml LB<br />
- 8µl amp<br />
- 10µl glycerol stock<br />
- Control pSecTag 2A in 8 ul of amp instead of cmp<br />
</p><br />
<p class="body_text"><br />
Glycerol stocks inoculated: 1.2, 1.6, 1.4, 1.18 (from second inoculation, in 4xcmp LB )<br />
All Batch I (from first inoculation, in 1xcmp for col.1-8 and 4xcmp for AA1 5xcmp) &Psectag 2A (Amp).<br />
</p><br />
<p class="body_text"><br />
Weiling: combined MMP-9 minipreps into one tube 11, 12, 13, 14, 17, 18, 1, 2, 4, 5, 6, 7, 10. This was labeled MMP9bbP00L, date, initials.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Nanodrop of pooled</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>MMP9 sample</td><br />
<td>NO DATA</td><br />
<td>NO DATA</td><br />
</tr><br />
<tr><br />
<td>MMP9 bb P00L</td><br />
<td>169.8</td><br />
<td>1.91</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 21st September</b><br />
</p><br />
<p class="body_text"><br />
Minipred-ed 10 ml LB 4x CMP ZEC BB Sample 1 (2z.1) and 6 (7z.2) (from second attempt of zeocin and backbone ligation)<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Tube no.</th><br />
<th>ng/ul</th><br />
<th>Absorption</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>3.2</td><br />
<td>0.087</td><br />
<td>2.41</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>2.9</td><br />
<td>0.068</td><br />
<td>2.51</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>20.1</td><br />
<td>1.249</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>21.1</td><br />
<td>0.590</td><br />
<td>1.48</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>8.3</td><br />
<td>0.186</td><br />
<td>1.20</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>8.4</td><br />
<td>0.178</td><br />
<td>1.32</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Pooled all 6 samples + Yanika’s minipreped sample IA+IB<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th></th><br />
<th>ng/ul</th><br />
<th>Abs</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>Sample</td><br />
<td>12.3</td><br />
<td>0.337</td><br />
<td>1.82</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculated 150µl of ZEC BB 1 into 4x cmp and 150 ml LB broth overnight.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 22nd September</b><br />
</p><br />
<p class="body_text"><br />
<a href="https://2013.igem.org/Team:UCL/Project/Protocols">Nanodrop</a> readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90<br />
</p><br />
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</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:41:34Z<p>AlexBates: </p>
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://2013.igem.org/File:KC_lab_2013.jpg');height:395px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/File:KC_lab_2013.jpgFile:KC lab 2013.jpg2013-10-05T03:40:39Z<p>AlexBates: uploaded a new version of &quot;File:KC lab 2013.jpg&quot;</p>
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<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:38:26Z<p>AlexBates: </p>
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/c/cc/Weiling_labs.jpg');height:435px;width:500px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/File:Weiling_labs.jpgFile:Weiling labs.jpg2013-10-05T03:37:25Z<p>AlexBates: </p>
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<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:35:32Z<p>AlexBates: </p>
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:500px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:31:30Z<p>AlexBates: </p>
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:30:39Z<p>AlexBates: Undo revision 316321 by AlexBates (talk)</p>
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<div class="gap"><br />
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T03:29:29Z<p>AlexBates: </p>
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<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<br />
</div><div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
</div><br />
</div><br />
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<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="body_text"><br />
<b>In the wet-lab we followed standard protocols with some of our own revisions. The details of our procedure are shown below. For an overview of what these procedures were used for, pleases see <a href="https://2013.igem.org/Team:UCL/Project/Experiments" target="_blank">experiments</a>.</b><br />
</p><br />
</div><div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">1.4% Agar</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Agar</td><br />
<td>7g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">5X M9 Salts</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>Na2HPO4</td><br />
<td>32g</td><br />
</tr><br />
<tr><br />
<td>KH2PO4</td><br />
<td>7.5g</td><br />
</tr><br />
<tr><br />
<td>NaCl</td><br />
<td>1.25g</td><br />
</tr><br />
<tr><br />
<td>NH4Cl</td><br />
<td>2.5g</td><br />
</tr><br />
<tr><br />
<td>RO HCL</td><br />
<td>500ml</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title"> 0.1M CaCl2/15% glycerol</p><br />
<p class="body_text"><br />
In a 50mL Falcon insert:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>100% Glycerol</td><br />
<td>7.5</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>37.5</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Minimal Agar</p><br />
<p class="body_text"><br />
Mix:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>5x M9 Salts</td><br />
<td>10mL</td><br />
</tr><br />
<tr><br />
<td>2 mg/ml Thiamine</td><br />
<td>50µl</td><br />
</tr><br />
<tr><br />
<td>20% D Glucose</td><br />
<td>1 mL</td><br />
</tr><br />
<tr><br />
<td>1M CaCl2</td><br />
<td>5µl</td><br />
</tr><br />
<tr><br />
<td>1M MgSO4</td><br />
<td>100µl</td><br />
</tr><br />
<tr><br />
<td>1.4% Agar</td><br />
<td>39 mL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">LB Media</p><br />
<p class="body_text"><br />
In 500mL Duran bottle insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Broth</td><br />
<td>10g</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>500 mL</td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Generating Competence Cells</p><br />
<p class="body_text"><br />
Locate a glycerol stock of untransformed E. coli, streak cells onto minimal agar plates and incubate at 37C for 16 hours.<br />
</p><br />
<p class="body_text"><br />
Once complete, pick a colony from the plate and place into a 50mL Falcon, containing 5mL LB & 100 uL 1M MgSO4 for 16 hours.<br />
</p><br />
<p class="body_text"><br />
After this, inoculate a 100mL shake flask with 1mL of culture from the Falcon tube. Take absorbance readings every 30 minutes until the absorbance reading is above 0.3. Once this is achieved, transfer the contents into two 50mL Falcon tubes and place on ice for 10 minutes. Perform centrifugation (~6,000 RPM) for 5 minutes and then resuspend in 10mL Calcium Chloride. Aliquot into eppendorf tubes (~500 uL per tube) and then store at very low temperatures (<-50C).<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4b/Streakingplatesigemucl2013.png');"></div><br />
<div class="description"><br />
<p class="minor_title">Streaking Plates</p><br />
<p class="body_text"><br />
Obtain agar plates (as many as required), streaking loops and cells to be streaked. Dip a streaking loop in the cell culture, and gently (so there is no damage to the agar) streak the loop onto the plate as described in the diagram below. Once finished, incubate at 37C overnight. <br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Plate generation (AMP, CMP & NoDrug)</p><br />
<p class="body_text"><br />
Heat up 50 mL of agar until molten (usually ~300 seconds using a 800W microwave). Douse in cold water to lower temperature. When still warm, but able to handle, it is possible to add an antibiotic drug for selection purposes (~50 uL). Once this complete, pour ~10mL into a petri dish and ensure that the whole surface is covered. Leave lid off for 30 minutes. Place lid on dish and then use, or store at ~5C.<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Glycerol Stock Generation</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity</th><br />
</tr><br />
<tr><br />
<td>LB Media</td><br />
<td>3mL</td><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>3 µl</td><br />
</tr><br />
<tr><br />
<td>Relevant Anti-bioitc</td><br />
<td>3 µl</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
12-16 hour 37C incubation. Insert into 1.5mL microcentrifuge tubes. Note absorbance. Add below, then store (-20C).<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µl)</th><br />
</tr><br />
<tr><br />
<td>Culture</td><br />
<td>500</td><br />
</tr><br />
<tr><br />
<td>Glycerol Stock</td><br />
<td>166</td><br />
</tr><br />
</table><br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">50X to 1X Dilution</p><br />
<p class="body_text"><br />
To a 1L Duran bottle, insert:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (mL)</th><br />
</tr><br />
<tr><br />
<td>50X TAE Buffer</td><br />
<td>20</td><br />
</tr><br />
<tr><br />
<td>RO H2O</td><br />
<td>980</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Transformation</p><br />
<p class="body_text"><br />
<b>1)</b> Remove one of your aliquots of competent cells from the -80C freezer and place onto ice.<br />
<b>2)</b> Add ~2uL of DNA to the competent cells. Leave in ice for ~45 minutes.<br />
<b>3)</b> Place tubes into 37C water bath for 10 minutes (heat shock).<br />
<b>4)</b> Place tubes into ice for 2 minutes.<br />
<b>5)</b> Add 1.3mL of Lb to the tubes and transfer all of the contents to new tubes. Incubate for 1 hour at 37C.<br />
<b>6)</b> Centrifuge at high RPM for 2 minutes. Discard the supernatant.<br />
<b>7)</b> Resuspend cell pellet into 100uL of LB.<br />
<b>8)</b> Spread contents onto petri dishes containing LB agar (may also contain antibiotic resistance for better selectivity.<br />
<b>9)</b> Incubate for 16 hours at 37C and then pick colonies if growth has occurred.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">Maxi/mini Preparation</p><br />
<p class="body_text"><br />
See <a href="http://www.google.co.uk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CDsQFjAA&url=http%3A%2F%2Fwww.qiagen.com%2Fresources%2FDownload.aspx%3Fid%3D%257B46205595-0440-459E-9D93-50EB02E5707E%257D%26lang%3Den%26ver%3D2&ei=-YpFUpvcF-yd0wWIw4HACg&usg=AFQjCNFGR5hl0QYv64lnVZDZWaw26BKA0A&sig2=JjaWz8EP2dxWJAMCpnLxCA&bvm=bv.53217764,d.d2k<br />
" target="_blank">protocol</a> for mini/maxi prep from Qiagen:<br />
</div><br />
<div class="description"><br />
<p class="minor_title">Analytical Digest</p><br />
<p class="body_text"><br />
Add the following items to a 1.5mL microcentrifuge tube and briefly (<10s) centrifuge to ensure all contents are mixed:<br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Required Quantity (µL)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>1.5g</td><br />
</tr><br />
<tr><br />
<td>Enzyme 1</td><br />
<td>150mL</td><br />
</tr><br />
<tr><br />
<td>Enzyme 2</td><br />
<td>3mL</td><br />
</tr><br />
</table><br />
</p><br />
<p class="body_text"><br />
Heat up the solution in a conical flask, until agarose has dissolved completely and the solution becomes clear. To the clear solution, add 2 ul of Ethidium Bromide and shake. Pour solution onto the the gel plate with the comb. Wait until gel has solidified after 20 minutes, the gel may now be ready for digest. <br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small" style="height:400px;"><br />
<div class="protocol"><br />
<p class="minor_title">Gel Electrophoreisis</p><br />
<p class="body_text"><br />
Add loading buffer to all samples (including laddder), then remove the comb from the solidified agarose gel, place the solidified agarose gel onto gel box and cover the gel box with 1x TAE buffer. Carefully load samples into the gel wells. Then cover the gel box with the lid. Run the gel on 120 Volts, 60 minutes condition. <br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/b/bd/Dongchanchoi.png');height:200px;width:321px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Nanodrop</p><br />
<p class="body_text"><br />
Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present. Open the Nanodrop program and the appropriate module (e.g., DNA). Wipe off the top and bottom sensors of the instrument with a Kimwipe. Pipette 1 μL of RO water onto the sensor. Bring down the lever arm. Follow the onscreen prompts to calibrate. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer EB or whatever solution your prep is in). Bring down the lever arm. Follow the onscreen prompts to blank. Wipe the sensors and pipette on 2 μL of your sample. Bring down the lever arm. Click Measure and record the concentration measured. For DNA, the peak should be at 260 nm, and as a general rule, the 260/280 ratio should be between 1.8 and 2.0. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary. Clean the sample surfaces once more after you are finished.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<p class="minor_title">PCR</p><br />
<p class="body_text"><br />
in a small 100uL reaction tube, add the following reagents:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Component</b></th><br />
<th>50ul Reaction</th><br />
<th>Final Concentration </th><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>to 50uL</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>5X NED Phusion buffer</td><br />
<td>10uL</td><br />
<td>1X</td><br />
</tr><br />
<tr><br />
<td>10mM dNTP</td><br />
<td>1ul</td><br />
<td>200uM</td><br />
<tr><br />
<td>10mM Forward Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>10mM Reverse Primer</td><br />
<td>2.5uL</td><br />
<td>0.5uM</td><br />
</tr><br />
<tr><br />
<td>Template DNA</td><br />
<td>2uL</td><br />
<td><250ng</td><br />
</tr><br />
<tr><br />
<td>DMSO (optional)</td><br />
<td>1.5uL</td><br />
<td>3%</td><br />
</tr><br />
<tr><br />
<td>Phusion DNA Polymerase</td><br />
<td>0.5uL</td><br />
<td>1.0 units</td><br />
</tr><br />
</tr><br />
</table><br />
<br />
<p class="body_text"><br />
Thermocycling conditions:<br />
</p><br />
<table><br />
<tr><br />
<th><b>Step</b></th><br />
<th>Temperature</th><br />
<th>Time </th><br />
</tr><br />
<tr><br />
<td>Initial Denaturation</td><br />
<td>98C</td><br />
<td>30s</td><br />
</tr><br />
<tr><br />
<td>25-35 cycles</td><br />
<td>98C<br />
<br />
45-72C<br />
<br />
72C</td><br />
<td>5-10s<br />
<br />
10-30s<br />
<br />
15-30s</td><br />
</tr><br />
<tr><br />
<td>Final Extension</td><br />
<td>72C</td><br />
<td>5-10min</td><br />
<tr><br />
<td>Hold</td><br />
<td>4-10C</td><br />
<td></td><br />
</tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</p><br />
<p class="body_text"><br />
In a 1.5mL eppendorf tube, add the following reagents, adds up to 10uL reaction volume<br />
</p><br />
<table><br />
<tr><br />
<th><b>Reagent</b></th><br />
<th>Volume</th><br />
</tr><br />
<tr><br />
<td>T4 Ligase</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>T4 ligase buffer</td><br />
<td>1uL</td><br />
</tr><br />
<tr><br />
<td>Insert</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>Backbone</td><br />
<td>2ul</td><br />
</tr><br />
<tr><br />
<td>H2O</td><br />
<td>4uL</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="gap"></div><br />
<p class="major_title">Mammalian Lab Protocols</p><br />
<div class="gap"></div><br />
<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/4/4f/Weiling_Labs.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Passaging Adherent Cells</p><br />
<p class="body_text"><br />
In order to keep cells healthy or increase stock, they must be sub-cultured - moving some cells from a previous culture into a new container with fresh growth medium. Here, we assume a 100mm dish. All solutions/equipment that come in contact with the cells must be sterile and work must be done in a laminar flow hood. <br />
<p class="body_text"><br />
<b>1)</b> Pipette spent medium and discard to waste.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Gently wash cells with PBS (5-10mL), then remove PBS to waste. Be careful not to disturb the cellular monolayer. This removes serum residue with trypsin inhibitors.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Add trypsin (2-5mL) to suspend cells. Ensure monolayer is covered. Incubate for 3-5 minutes at 37C. <br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>NOTE:</b> Care should be taken to<br />
avoid leaving cells exposed to the trypsin<br />
longerthan necessary. Care should also be<br />
taken that the cells not be forced to detach<br />
prematurely, as this may result in clumping.<br />
</p><br />
<p class="body_text"><br />
<b>4)</b>Add serum-containing medium(10mL) and pipette the cells up and<br />
down until the cells are dispersed into<br />
a single cells suspension. <br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Add the appropriate volume of cell<br />
suspension (dependent on confluence/cell count - generally for 100% confluence split 1:4) to a new flask/dish containing medium (end volume 10mL).<br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Place dish(es) in incubator at 37C. Leave for 3-4 days before next passage. <br />
</p><br />
</div><br />
</div><br />
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<br />
<div class="row_small"><br />
<div class="protocol"><br />
<div class="protocol" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a5/KC_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Stable Transfection Of Adherent Cells</p><br />
<p class="body_text"><br />
For the stable transfection of eukaryotic adherent cell types in a single well of a 6-well plate. When transfecting multiple wells, make a 'master mix' with 110% of all solutions.<br />
<p class="body_text"><br />
<b>1)</b> The day before transfection, seed 0.9-4x10^5 cell per well of the six well plate with 2ml of appropriate growth medium. This should produce a confluence of 40-80% for the next day's transfection.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
<b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.<br />
</p><br />
<p class="body_text"><br />
<b>5)</b> Incubate at room temperature for 5-10mins to allow for complex formation.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> Meanwhile, gently aspirate growth medium from dish and wash cells with 3ml. <br />
</p><br />
<p class="body_text"><br />
<b>7)</b> Add 600µl of cell growth medium (with serum and antibiotics)to reaction tube. Mix up and down with pipette and immediately transfer total volume to well. <br />
</p><br />
<p class="body_text"><br />
<b>8)</b> Change medium and wash with PBS. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Incubate for 24-48 hours. <br />
</p><br />
<p class="body_text"><br />
<b>9)</b> Assay for gene expression. <br />
</p><br />
</div><br />
</div><br />
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<div class="row_small"><br />
<div class="protocol"><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/16/ALex_Bates_lab_2013.jpg');height:316px;width:400px"></div><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Amyloid Degradation Assay</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
<p class="body_text"><br />
Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.<br />
</p><br />
<p class="body_text"><br />
To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
<div class="full_row"><br />
<p class="body_text"><br />
a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. <br />
</p><br />
<p class="body_text"><br />
b. For fAβ digestions, 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days.<br />
</p><br />
<p class="body_text"><br />
c. After digestion, analyse the reaction by Congo red assay.<br />
</p><br />
<p class="body_text"><br />
</div><br />
</div><br />
<div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><div class="gap"></div><br />
<div class="row_small"><br />
<div class="protocol"></div><br />
<div class="description"><br />
<p class="minor_title">Congo Red Spectrophotometric assay </p><br />
<p class="body_text"><br />
<b>1)</b> Make up a 7 mg/mL solution of Congo Red in a buffer solution of 5mM potassium phosphate, 150mM NaCl (pH7.4). Filter through a 0.2µm syringe immediately before using.<br />
</p><br />
<p class="body_text"><br />
<b>2)</b> At room temperature zero a UV–Vis spectrophotometer between 400 and700 nm with a disposable cuvette containing 1mL phosphate buffer.<br />
</p><br />
<p class="body_text"><br />
<b>3)</b> To the the phosphate buffer, add 5µL of the Congo Red solution. Scan between 400 and 700 nm and take a record of the spectrum.<br />
</p><br />
</p><br />
<p class="body_text"><br />
</p><br />
<p class="body_text"><br />
<b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.<br />
</p><br />
</div><br />
<div class="gap"></div><br />
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<p class="body_text"><br />
<b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.<br />
</p><br />
<p class="body_text"><br />
<b>6)</b> For a microscopic analysis, transfer the protein/lysate-Congo Red solution to a centrifuge tube. Centrifuge (12,000–14,000 rpm) to pellet the fibrils. Wash the fibrils with water, resuspend the fibrils in a small amount of water, and place on a microscope slide. Let the sample dry in air and analyse under polarized light. If transfection with MMP-9 has been successful, the assay should not be strongly indicative of fibrils.<br />
</p><br />
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<p class="major_title">ZEOCIN RESISTANCE</p><br />
<p class="minor_title">For Selecting Transfected Cells</p><br />
<p class="body_text"><br />
To easily select cells that were transfected with our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a>, we required a selectable marker that would work in all of our <a href="https://2013.igem.org/Team:UCL/Project/Chassis" target="_blank">chassis</a>, particularly HeLa cells and microglia, and would enable us to easily eliminate cells that have not taken up our recombinant plasmid. Zeocin is a widely used glycopeptide antibiotic, a formulation of phleomycin D1. It is capable of binding to and cleaving DNA, leading to cell necrosis in both eukaryotes and aerobic prokaryotes. Commonly outside of cells, in copper-chelated form, zeocin is inactive. When zeocin enters a cell, the Cu2+, which makes it appear blue, is reduced to Cu+ and then removed, activating zeocin, which then intercalates into DNA <a href="http://tools.invitrogen.com/content/sfs/manuals/zeocin_man.pdf" target="_blank">(Invitrogen)</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
A 375 base pair bacterial gene encodes the Streptoalloteichus hindustanus bleomycin resistance protein (She ble protein). The She ble protein in mammalian cells is predominantly localised at the nucleus, specifically at euchromatin <a href="http://www.ncbi.nlm.nih.gov/pubmed/7505390" target="_blank">(Calmels et al. 199)</a>. This small protein that has a strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA. However, the protection it confers, in human cells at least, while considerable, is not complete. However, it is an extremely useful selectable marker, that will be invaluable to the iGEM registry <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>.<br />
<p class="body_text"><br />
In order to establish that this BioBrick worked, we had to first determine Zeocin’s killing efficacy against HeLa cells by creating a kill curve. <br />
</p> <br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Creating The BioBrick</p><br />
</p><br />
<p class="body_text"><br />
In order for mammalian cells to express Zeocin resistance, our Zeocin resistance biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> includes a CMV promoter.</p><br />
</p><br />
<p class="body_text"><br />
This biobrick benefits the iGEM Registry tremendously by providing a suitable selectable marker for cell culture and mammalian transfection, which was previously non-existent in the iGEM Registry. This biobrick will make mammalian transfection easier and will encourage iGEM teams to venture more into mammalian synthetic biology.<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<p class="major_title">EXPERIMENTS AND RESULTS</p><br />
<div class="gap"></div><br />
<br />
<br />
<div class="full_page"><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d8/LogHeLagrowthcurve.png');height:500px;width:740px"></div><br />
<p class="minor_title">Growth Curve</p><br />
<p class="body_text"><br />
Before using HeLa cells for transfection and characterisation, we carried out basic characterisation of the chassis. For this, we conducted a HeLa growth curve.<br />
</p><br />
<p class="body_text"><br />
There is an exponential growth until the 4th day. After the 4th day, the growth of HeLa cells slows down. Some cells start to detach and die from over-confluency.<br />
</p><br />
<p class="body_text"><br />
Through this graph, we were able to decide that we would split or passage HeLa cells every 3 to 4 days to maintain good cell health. Maintaining good cell confluence is important for maximum transfection efficiency.<br />
</p><br />
<p class="body_text"><br />
The ideal HeLa confluency for transfection is 70%. Hence, from this graph, we can plan to split the cells one day before transfection to attain 70% confluency.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/d/dc/Zeo_kill_curve_healthy_HeLa.png');height:500px;width:740px"></div><br />
<p class="minor_title">Zeocin Kill Curve</p><br />
<br />
</p><br />
<p class="body_text"><br />
In order to determine the concentrations of Zeocin at which HeLa cells start to die, we carried out a Zeocin Kill curve.<br />
</p><br />
<p class="body_text"><br />
This helped us to decide the concentrations of Zeocin we would use to characterise our Zeocin resistance Biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a>.<br />
</p><br />
<p class="body_text"><br />
From this data, we hypothesised that if our HeLa cells survive in 50-200 ug/mL of Zeocin, our Biobrick is successful.<br />
</p><br />
<p class="body_text"><br />
By carrying out this Kill Curve, we were able to observe the appearance of healthy cells versus swollen and dead cells for objective characterisation of our Zeocin resistance biobrick.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="minor_title">Characterisation</p><br />
<p class="body_text"> <br />
In order to characterise our new BioBrick, we seeded the wells of a six-well plate with HeLa cells at passage 20 and transfected all but one (the control) of these wells with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> using SuperFect transfection reagent. We used three six-well plates, exposed one to 50 ug/ml zeocin, another to 100 ug/ml zeocin and the last to 150 ug/ml Zeocin. These concentrations were chosen based on our kill curve data. We plotted the percentage of cells we deemed viable in our plates in a blind study over eight days. Viability was assessed visually by noting the surface area occupied by bloated/dead cells versus healthier cells. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/4/41/Zeocin_50.png');height:293px;width:478px;"></div><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/1b/Zeocin_100.png');height:293px;width:478px"></div><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/7/74/Graph_150_Zec.png');height:293px;width:488px"></div><br />
</p><br />
<p class="body_text"> <br />
Our graphs clearly demonstrate that the transfected HeLa wells fared better than the non-transfected ones, by a margin of about 10%. This is to be expected, for three reasons:<br />
</p><br />
<p class="body_text"> <br />
Firstly, the sample of <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> was impure and so not all of the transfected cells will have been transfected with She ble. Moreover, we expect only about 40% of the cells to have been transfected at all. This is a conservative estimate based on the fact that we have not worked with mammalian cells or done a transfection before iGEM. <br />
</p><br />
<p class="body_text"> <br />
Secondly, viability readings are distorted by the fact that HeLa cells die due to over confluence (population stress and lack of nutrients) meaning that, especially in the transfected wells, not all cell death was due to zeocin action. For more information, see the box below. <br />
</p><br />
<p class="body_text"> <br />
Thirdly, the <I>she ble</I> gene does not confer full immunity to transfected cells, only a degree of resistance, which is why the transfected cells die quicker at higher zeocin concentrations. All of the graphs show the transfected cells experiencing a slight rise in viability at the 6-7th day. We hypothesise that this is because zeocin resistant cell clusters expand into the space left by unsuccessfully transfected HeLa cells that have died. The curve continues its downward trend, however, becaase of over confluence. <br />
</p><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<div class="gap"></div><br />
<div class="gap"></div><br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="minor_title">Types of Cell Death</p><br />
<p class="body_text"> <br />
<b>Above.</b> Cell death due to over confluence in our HeLa cells transfected with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> in 200 ug/ml of Zeocin, after four days.<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/3/33/Over_confluent_cells_2013_copy.png');height:300px;width:400px"></div> <br />
<p class="body_text"><br />
<b>Below.</b> Cell death due to zeocin in our non-transfected HeLa cells in 200 ug/ml of zeocin, after four days.<br />
</p><br />
<div class="gap"></div><br />
<p class="body_text"><br />
In both our transfected cell wells and in the control there is net cell death over time. Though it is clear from the above graphs that the transfected cells fair better over time, it is important to note that the cell death these wells sustained was proportionally more due to over confluence than due to Zeocin. We ran a set of transfected wells and a control of non transfected HeLa cells over three days, and took a set of images, two of which are shown to the right. The upper image shows an over confluent dish of cells, in which the viability is low because cells are dying due to population stress and lack of nutrients. The swollen, stretched, spindly HeLa cells in the lower image are characteristic of Zeocin imposed cell death; they have lysed or are ready to lyse. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/74/Zeo_splat_HeLa_copy.png');height:300px;width:400px"></div><br />
<p class="body_text"><br />
To show this quantitatively, we used a Vi-Cell (cell viability analyser), which identifies dead cells using tryphan blue and measures cell diameter, to take readings from a sample of healthy cells, our control cells at 200 ug/ml Zeocin and a well of transfected cells at 200 ug/ml Zeocin.<br />
<table><br />
<tr><br />
<th><b>Sample</b></th><br />
<th>Average Diameter (um)</th><br />
</tr><br />
<tr><br />
<td>Healthy Cells</td><br />
<td>13.18</td><br />
</tr><br />
<tr><br />
<td>Non-transfected HeLa cells</td><br />
<td>16.85</td><br />
</tr><br />
<tr><br />
<td>Transfected HeLa cells</td><br />
<td>14.54</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
The non-transfected cells susceptible to Zeocin are clearly swollen, while the swelling in transfected cells is substantially less. Again, this is because the <I>she ble</I> gene does not confer full Zeocin immunity, but Zeocin resistance <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>. Visually, down the microscope, we observed that the 'control' cells would swell to up to about 20 um in diameter, small clusters of smaller diameter, healthier cells. These clusters were more common in the transfected HeLa wells, assumedly where surviving transfected cells have proliferated into gaps left by their non-transfected neighbours dying.<br />
</p> <br />
<p class="body_text"><br />
The photo to the right clearly shows that the control has less viable cells than the transfected well. This photo is of the 150 ug/ml zeocin at day eight. The growth medium in the control is far pinker than in the other wells. This is because live cells use of nutrients in he growth medium and sap its colour.<br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d5/Six-well-plate-2013.jpg');height:375px;width:500px"></div> <br />
</p><br />
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<p class="major_title">ZEOCIN RESISTANCE</p><br />
<p class="minor_title">For Selecting Transfected Cells</p><br />
<p class="body_text"><br />
To easily select cells that were transfected with our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a>, we required a selectable marker that would work in all of our <a href="https://2013.igem.org/Team:UCL/Project/Chassis" target="_blank">chassis</a>, particularly HeLa cells and microglia, and would enable us to easily eliminate cells that have not taken up our recombinant plasmid. Zeocin is a widely used glycopeptide antibiotic, a formulation of phleomycin D1. It is capable of binding to and cleaving DNA, leading to cell necrosis in both eukaryotes and aerobic prokaryotes. Commonly outside of cells, in copper-chelated form, zeocin is inactive. When zeocin enters a cell, the Cu2+, which makes it appear blue, is reduced to Cu+ and then removed, activating zeocin, which then intercalates into DNA <a href="http://tools.invitrogen.com/content/sfs/manuals/zeocin_man.pdf" target="_blank">(Invitrogen)</a>.<br />
</p><br />
<br />
<p class="body_text"><br />
A 375 base pair bacterial gene encodes the Streptoalloteichus hindustanus bleomycin resistance protein (She ble protein). The She ble protein in mammalian cells is predominantly localised at the nucleus, specifically at euchromatin <a href="http://www.ncbi.nlm.nih.gov/pubmed/7505390" target="_blank">(Calmels et al. 199)</a>. This small protein that has a strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA. However, the protection it confers, in human cells at least, while considerable, is not complete. However, it is an extremely useful selectable marker, that will be invaluable to the iGEM registry <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>.<br />
<p class="body_text"><br />
In order to establish that this BioBrick worked, we had to first determine Zeocin’s killing efficacy against HeLa cells by creating a kill curve. <br />
</p> <br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Creating The BioBrick</p><br />
</p><br />
<p class="body_text"><br />
In order for mammalian cells to express Zeocin resistance, our Zeocin resistance biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> includes a CMV promoter.</p><br />
</p><br />
<p class="body_text"><br />
This biobrick benefits the iGEM Registry tremendously by providing a suitable selectable marker for cell culture and mammalian transfection, which was previously non-existent in the iGEM Registry. This biobrick will make mammalian transfection easier and will encourage iGEM teams to venture more into mammalian synthetic biology.<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
<p class="major_title">EXPERIMENTS AND RESULTS</p><br />
<div class="gap"></div><br />
<br />
<br />
<div class="full_page"><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d8/LogHeLagrowthcurve.png');height:500px;width:740px"></div><br />
<p class="minor_title">Growth Curve</p><br />
<p class="body_text"><br />
Before using HeLa cells for transfection and characterisation, we carried out basic characterisation of the chassis. For this, we conducted a HeLa growth curve.<br />
</p><br />
<p class="body_text"><br />
There is an exponential growth until the 4th day. After the 4th day, the growth of HeLa cells slows down. Some cells start to detach and die from over-confluency.<br />
</p><br />
<p class="body_text"><br />
Through this graph, we were able to decide that we would split or passage HeLa cells every 3 to 4 days to maintain good cell health. Maintaining good cell confluence is important for maximum transfection efficiency.<br />
</p><br />
<p class="body_text"><br />
The ideal HeLa confluency for transfection is 70%. Hence, from this graph, we can plan to split the cells one day before transfection to attain 70% confluency.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/d/dc/Zeo_kill_curve_healthy_HeLa.png');height:500px;width:740px"></div><br />
<p class="minor_title">Zeocin Kill Curve</p><br />
<br />
</p><br />
<p class="body_text"><br />
In order to determine the concentrations of Zeocin at which HeLa cells start to die, we carried out a Zeocin Kill curve.<br />
</p><br />
<p class="body_text"><br />
This helped us to decide the concentrations of Zeocin we would use to characterise our Zeocin resistance Biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a>.<br />
</p><br />
<p class="body_text"><br />
From this data, we hypothesised that if our HeLa cells survive in 50-200 ug/mL of Zeocin, our Biobrick is successful.<br />
</p><br />
<p class="body_text"><br />
By carrying out this Kill Curve, we were able to observe the appearance of healthy cells versus swollen and dead cells for objective characterisation of our Zeocin resistance biobrick.<br />
</p><br />
</div><br />
<br />
<div class="gap"></div><br />
<br />
<div class="full_page"><br />
<p class="minor_title">Characterisation</p><br />
<p class="body_text"> <br />
In order to characterise our new BioBrick, we seeded the wells of a six-well plate with HeLa cells at passage 20 and transfected all but one (the control) of these wells with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> using SuperFect transfection reagent. We used three six-well plates, exposed one to 50 ug/ml zeocin, another to 100 ug/ml zeocin and the last to 150 ug/ml Zeocin. These concentrations were chosen based on our kill curve data. We plotted the percentage of cells we deemed viable in our plates in a blind study over eight days. Viability was assessed visually by noting the surface area occupied by bloated/dead cells versus healthier cells. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/4/41/Zeocin_50.png');height:293px;width:478px;"></div><br />
<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/1/1b/Zeocin_100.png');height:293px;width:478px"></div><br />
<div class="main_image" style="background-image:url('https://static.igem.org/mediawiki/2013/7/74/Graph_150_Zec.png');height:293px;width:488px"></div><br />
</p><br />
<p class="body_text"> <br />
Our graphs clearly demonstrate that the transfected HeLa wells fared better than the non-transfected ones, by a margin of about 10%. This is to be expected, for three reasons:<br />
</p><br />
<p class="body_text"> <br />
Firstly, the sample of <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> was impure and so not all of the transfected cells will have been transfected with She ble. Moreover, we expect only about 40% of the cells to have been transfected at all. This is a conservative estimate based on the fact that we have not worked with mammalian cells or done a transfection before iGEM. <br />
</p><br />
<p class="body_text"> <br />
Secondly, viability readings are distorted by the fact that HeLa cells die due to over confluence (population stress and lack of nutrients) meaning that, especially in the transfected wells, not all cell death was due to zeocin action. For more information, see the box below. <br />
</p><br />
<p class="body_text"> <br />
Thirdly, the <I>she ble</I> gene does not confer full immunity to transfected cells, only a degree of resistance, which is why the transfected cells die quicker at higher zeocin concentrations. All of the graphs show the transfected cells experiencing a slight rise in viability at the 6-7th day. We hypothesise that this is because zeocin resistant cell clusters expand into the space left by unsuccessfully transfected HeLa cells that have died. The curve continues its downward trend, however, becaase of over confluence. <br />
</p><br />
</div><br />
<br />
<br />
<div class="gap"></div><br />
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<p class="body_text"> <br />
<b>Above.</b> Cell death due to over confluence in our HeLa cells transfected with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> in 200 ug/ml of Zeocin, after four days.<br />
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<b>Below.</b> Cell death due to zeocin in our non-transfected HeLa cells in 200 ug/ml of zeocin, after four days.<br />
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In both our transfected cell wells and in the control there is net cell death over time. Though it is clear from the above graphs that the transfected cells fair better over time, it is important to note that the cell death these wells sustained was proportionally more due to over confluence than due to Zeocin. We ran a set of transfected wells and a control of non transfected HeLa cells over three days, and took a set of images, two of which are shown to the right. The upper image shows an over confluent dish of cells, in which the viability is low because cells are dying due to population stress and lack of nutrients. The swollen, stretched, spindly HeLa cells in the lower image are characteristic of Zeocin imposed cell death; they have lysed or are ready to lyse. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/74/Zeo_splat_HeLa_copy.png');height:300px;width:400px"></div><br />
<p class="body_text"><br />
To show this quantitatively, we used a Vi-Cell (cell viability analyser), which identifies dead cells using tryphan blue and measures cell diameter, to take readings from a sample of healthy cells, our control cells at 200 ug/ml Zeocin and a well of transfected cells at 200 ug/ml Zeocin.<br />
<table><br />
<tr><br />
<th><b>Sample</b></th><br />
<th>Average Diameter (um)</th><br />
</tr><br />
<tr><br />
<td>Healthy Cells</td><br />
<td>13.18</td><br />
</tr><br />
<tr><br />
<td>Non-transfected HeLa cells</td><br />
<td>16.85</td><br />
</tr><br />
<tr><br />
<td>Transfected HeLa cells</td><br />
<td>14.54</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
The non-transfected cells susceptible to Zeocin are clearly swollen, while the swelling in transfected cells is substantially less. Again, this is because the <I>she ble</I> gene does not confer full Zeocin immunity, but Zeocin resistance <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>. Visually, down the microscope, we observed that the 'control' cells would swell to up to about 20 um in diameter, small clusters of smaller diameter, healthier cells. These clusters were more common in the transfected HeLa wells, assumedly where surviving transfected cells have proliferated into gaps left by their non-transfected neighbours dying.<br />
</p><br />
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<p class="body_text"><br />
The photo to the right clearly shows that the control has less viable cells than the transfected well. This photo is of the 150 ug/ml zeocin at day eight. The growth medium in the control is far pinker than in the other wells. This is because live cells use of nutrients in he growth medium and sap its colour. <br />
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<p class="major_title">ZEOCIN RESISTANCE</p><br />
<p class="minor_title">For Selecting Transfected Cells</p><br />
<p class="body_text"><br />
To easily select cells that were transfected with our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a>, we required a selectable marker that would work in all of our <a href="https://2013.igem.org/Team:UCL/Project/Chassis" target="_blank">chassis</a>, particularly HeLa cells and microglia, and would enable us to easily eliminate cells that have not taken up our recombinant plasmid. Zeocin is a widely used glycopeptide antibiotic, a formulation of phleomycin D1. It is capable of binding to and cleaving DNA, leading to cell necrosis in both eukaryotes and aerobic prokaryotes. Commonly outside of cells, in copper-chelated form, zeocin is inactive. When zeocin enters a cell, the Cu2+, which makes it appear blue, is reduced to Cu+ and then removed, activating zeocin, which then intercalates into DNA <a href="http://tools.invitrogen.com/content/sfs/manuals/zeocin_man.pdf" target="_blank">(Invitrogen)</a>.<br />
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A 375 base pair bacterial gene encodes the Streptoalloteichus hindustanus bleomycin resistance protein (She ble protein). The She ble protein in mammalian cells is predominantly localised at the nucleus, specifically at euchromatin <a href="http://www.ncbi.nlm.nih.gov/pubmed/7505390" target="_blank">(Calmels et al. 199)</a>. This small protein that has a strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA. However, the protection it confers, in human cells at least, while considerable, is not complete. However, it is an extremely useful selectable marker, that will be invaluable to the iGEM registry <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>.<br />
<p class="body_text"><br />
In order to establish that this BioBrick worked, we had to first determine Zeocin’s killing efficacy against HeLa cells by creating a kill curve. <br />
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<p class="minor_title">Creating The BioBrick</p><br />
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In order for mammalian cells to express Zeocin resistance, our Zeocin resistance biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> includes a CMV promoter.</p><br />
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<p class="body_text"><br />
This biobrick benefits the iGEM Registry tremendously by providing a suitable selectable marker for cell culture and mammalian transfection, which was previously non-existent in the iGEM Registry. This biobrick will make mammalian transfection easier and will encourage iGEM teams to venture more into mammalian synthetic biology.<br />
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<p class="major_title">EXPERIMENTS AND RESULTS</p><br />
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<p class="minor_title">Growth Curve</p><br />
<p class="body_text"><br />
Before using HeLa cells for transfection and characterisation, we carried out basic characterisation of the chassis. For this, we conducted a HeLa growth curve.<br />
</p><br />
<p class="body_text"><br />
There is an exponential growth until the 4th day. After the 4th day, the growth of HeLa cells slows down. Some cells start to detach and die from over-confluency.<br />
</p><br />
<p class="body_text"><br />
Through this graph, we were able to decide that we would split or passage HeLa cells every 3 to 4 days to maintain good cell health. Maintaining good cell confluence is important for maximum transfection efficiency.<br />
</p><br />
<p class="body_text"><br />
The ideal HeLa confluency for transfection is 70%. Hence, from this graph, we can plan to split the cells one day before transfection to attain 70% confluency.<br />
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<p class="minor_title">Zeocin Kill Curve</p><br />
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</p><br />
<p class="body_text"><br />
In order to determine the concentrations of Zeocin at which HeLa cells start to die, we carried out a Zeocin Kill curve.<br />
</p><br />
<p class="body_text"><br />
This helped us to decide the concentrations of Zeocin we would use to characterise our Zeocin resistance Biobrick <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a>.<br />
</p><br />
<p class="body_text"><br />
From this data, we hypothesised that if our HeLa cells survive in 50-200 ug/mL of Zeocin, our Biobrick is successful.<br />
</p><br />
<p class="body_text"><br />
By carrying out this Kill Curve, we were able to observe the appearance of healthy cells versus swollen and dead cells for objective characterisation of our Zeocin resistance biobrick.<br />
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<p class="minor_title">Characterisation</p><br />
<p class="body_text"> <br />
In order to characterise our new BioBrick, we seeded the wells of a six-well plate with HeLa cells at passage 20 and transfected all but one (the control) of these wells with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> using SuperFect transfection reagent. We used three six-well plates, exposed one to 50 ug/ml zeocin, another to 100 ug/ml zeocin and the last to 150 ug/ml Zeocin. These concentrations were chosen based on our kill curve data. We plotted the percentage of cells we deemed viable in our plates in a blind study over eight days. Viability was assessed visually by noting the surface area occupied by bloated/dead cells versus healthier cells. <br />
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<p class="body_text"> <br />
Our graphs clearly demonstrate that the transfected HeLa wells fared better than the non-transfected ones, by a margin of about 10%. This is to be expected, for three reasons:<br />
</p><br />
<p class="body_text"> <br />
Firstly, the sample of <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> was impure and so not all of the transfected cells will have been transfected with She ble. Moreover, we expect only about 40% of the cells to have been transfected at all. This is a conservative estimate based on the fact that we have not worked with mammalian cells or done a transfection before iGEM. <br />
</p><br />
<p class="body_text"> <br />
Secondly, viability readings are distorted by the fact that HeLa cells die due to over confluence (population stress and lack of nutrients) meaning that, especially in the transfected wells, not all cell death was due to zeocin action. For more information, see the box below. <br />
</p><br />
<p class="body_text"> <br />
Thirdly, the <I>she ble</I> gene does not confer full immunity to transfected cells, only a degree of resistance, which is why the transfected cells die quicker at higher zeocin concentrations. All of the graphs show the transfected cells experiencing a slight rise in viability at the 6-7th day. We hypothesise that this is because zeocin resistant cell clusters expand into the space left by unsuccessfully transfected HeLa cells that have died. The curve continues its downward trend, however, becaase of over confluence. <br />
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<p class="minor_title">Types of Cell Death</p><br />
<p class="body_text"> <br />
<b>Above.</b> Cell death due to over confluence in our HeLa cells transfected with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018001)</a> in 200 ug/ml of Zeocin, after four days.<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/3/33/Over_confluent_cells_2013_copy.png');height:300px;width:400px"></div> <br />
<p class="body_text"><br />
<b>Below.</b> Cell death due to zeocin in our non-transfected HeLa cells in 200 ug/ml of zeocin, after four days.<br />
</p><br />
<div class="gap"></div><br />
<p class="body_text"><br />
In both our transfected cell wells and in the control there is net cell death over time. Though it is clear from the above graphs that the transfected cells fair better over time, it is important to note that the cell death these wells sustained was proportionally more due to over confluence than due to Zeocin. We ran a set of transfected wells and a control of non transfected HeLa cells over three days, and took a set of images, two of which are shown to the right. The upper image shows an over confluent dish of cells, in which the viability is low because cells are dying due to population stress and lack of nutrients. The swollen, stretched, spindly HeLa cells in the lower image are characteristic of Zeocin imposed cell death; they have lysed or are ready to lyse. <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/74/Zeo_splat_HeLa_copy.png');height:300px;width:400px"></div><br />
<p class="body_text"><br />
To show this quantitatively, we used a Vi-Cell (cell viability analyser), which identifies dead cells using tryphan blue and measures cell diameter, to take readings from a sample of healthy cells, our control cells at 200 ug/ml Zeocin and a well of transfected cells at 200 ug/ml Zeocin.<br />
<table><br />
<tr><br />
<th><b>Sample</b></th><br />
<th>Average Diameter (um)</th><br />
</tr><br />
<tr><br />
<td>Healthy Cells</td><br />
<td>13.18</td><br />
</tr><br />
<tr><br />
<td>Non-transfected HeLa cells</td><br />
<td>16.85</td><br />
</tr><br />
<tr><br />
<td>Transfected HeLa cells</td><br />
<td>14.54</td><br />
</tr><br />
</table><br />
<p class="body_text"><br />
The non-transfected cells susceptible to Zeocin are clearly swollen, while the swelling in transfected cells is substantially less. Again, this is because the <I>she ble</I> gene does not confer full Zeocin immunity, but Zeocin resistance <a href="http://www.ncbi.nlm.nih.gov/pubmed/15755800" target="_blank">(Oliva-Trastoy 2005)</a>. Visually, down the microscope, we observed that the 'control' cells would swell to up to about 20 um in diameter, small clusters of smaller diameter, healthier cells. These clusters were more common in the transfected HeLa wells, assumedly where surviving transfected cells have proliferated into gaps left by their non-transfected neighbours dying.<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d5/Six-well-plate-2013.jpg<br />
');height:375px;width:500px"></div> <br />
<p class="body_text"><br />
The photo to the right clearly shows that the control has less viable cells than the transfected well. This photo is of the 150 ug/ml zeocin at day eight. The growth medium in the control is far pinker than in the other wells. This is because live cells use of nutrients in he growth medium and sap its colour. <br />
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<div></div>AlexBateshttp://2013.igem.org/File:IMG-20131004-00191.jpgFile:IMG-20131004-00191.jpg2013-10-05T03:06:26Z<p>AlexBates: uploaded a new version of &quot;File:IMG-20131004-00191.jpg&quot;</p>
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<div></div>AlexBateshttp://2013.igem.org/File:IMG-20131004-00191.jpgFile:IMG-20131004-00191.jpg2013-10-05T03:04:20Z<p>AlexBates: </p>
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<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T02:54:36Z<p>AlexBates: </p>
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<p class="minor_title">Dr. Darren Nesbeth</p> <br />
<p class="body_text"><b>Lecturer in Synthetic and Molecular Biology.</b> Supervisor and overall co-ordinator of iGEM at UCL. Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning.</p><br />
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<p class="minor_title">Philipp Boeing</p> <br />
<p class="body_text"><b>Msc Computer Science. Human Practice Supervisor.</b> I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!<br />
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<p class="minor_title">Yanika Borg</p> <br />
<p class="body_text"><b> PhD Student. Bacterial Lab Supervisor.</b> When I’m not working on my PhD in Synthetic Biology, I am supervising Spotless Mind’s bacterial team. My role is to oversee all experiments carried out on E. coli, to demonstrate molecular cloning techniques to the team, and to calm Andy down on a daily basis. This is my second year supervising iGEM at UCL, and I love the whole experience.</p><br />
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<p class="minor_title">Alex Kinna</p> <br />
<p class="body_text"><b>PhD Student. Mammalian Lab Supervisor.</b> I am a 2nd year PhD student studying biochemical and protein engineering. My role is to advise and support mammalian cell culture, testing of circuits in mammalian cells and production of target proteins.<br />
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<div class="col_1"><p class="body_text"><b>In the course of the development of our idea, we consulted synthetic biologists, neuroscientists, neurosurgeons, psychiatrists and geneticists and took on board their feedback in order to develop our idea and add the detail to our genetic circuit. We show what advice we received here and how this advice was incorporated into our final project.</p></b><br />
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<p class="body_text"><b>Dr. Jeremy Cook</b> is a senior lecturer and the programme tutor for the Neuroscience Bsc at UCL. His research interests concern the development the visual system, including the embryonic emergence of retinal cell patterns. He advised us to carefully consider the neurosurgical implications of our project, noting the preferability of an autograph of microglia, and the need to design our circuit so that the microglia only become de-activated at plaques, because a degree of activation is required for chemotaxis. <br />
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<p class="body_text"><b>Dr. John Scholes</b> is an honorary senior lecturer, and lectures on the Neuroscience Bsc course at UCL. He supported the idea of using BDNF in the circuit in order to stop cell cycle re-entry in AD and suggested ApoE as a possible circuit component, as it could increase the activity of our chosen protease. <br />
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<div class="col_2"><p class="body_text"><b>Dr. Jennifer Pococks'</b> research involves cell signaling in neurodegenerative dieseases and this onvolves the study of microglia in the context of AD. She advised the team on using microglia in the lab.<br />
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<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>Professor Patrick Haggard</b> is a prominent figure in neuroethical debate, Patrick Haggard is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, UCL. We are thankful to him for providing inspiration and being a sounding board for our neuroethical investigations, and for agreeing to be filmed as part of our documentary. <br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor John Powell</b> is a geneticist in the Department of Neuroscience and Psychological Medicine at Kings College London. His research interests are in the application of human genetics to the study of neurological and psychiatric disorders; in schizophrenia and autism. He helped direct our theoretical work on how synthetic neurobiology could be expanded to different brain conditions, therapies and enhancements.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor Stephen Hart</b> works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We are thankful for him on his advice concerning how to transfect native microglia in vivo. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using lipid-peptide nanocomplexes. This result of his was un-expected as his team had been trying to transfect cancerous cells in rat brains but increases the feasibility of our idea.<br />
<br />
<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Dr. Tammy Cheng</b> and <b>Dr. Paul Bates</b> are scientists at the BMM lab at Cancer Research UK that envisioned and helped team member Alex create and run a bioinformatics network analysis programme, as well serving as discussing our ideas more generally and so helping to improve them. <br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="major_title">OTHER ACKNOWLEDGMENTS</p><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>content</b></p><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>content</b><br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><p class="body_text"><b>Lubmilla Ruban</b> is a stem cell biologist in UCL's Biochemical Engineering Department, who manages the Cell Culture, Cell Bioprocessing and the Liquid Nitrogen facilities. We are thankful to her for teaching us the basics of mammalian cell culture, including how to passage cells and how to use the essential equipment of the mammalian labs. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Sean Tuite</b> is a first year undergraduate film student to whom we owe thanks for his help in creating our documentary as cameraman and editor.<br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
</div><br />
<br />
</div><br />
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<script type="text/javascript" src="https://2013.igem.org/Team:UCL/static/footer.js?action=raw&ctype=text/javascript"> <br />
</script><br />
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</body><br />
</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T02:50:14Z<p>AlexBates: </p>
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<p class="major_title">SUPERVISORS</p><br />
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<div class="col_1"><br />
<br />
<p class="minor_title">Dr. Darren Nesbeth</p> <br />
<p class="body_text"><b>Lecturer in Synthetic and Molecular Biology.</b> Supervisor and overall co-ordinator of iGEM at UCL. Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Philipp Boeing</p> <br />
<p class="body_text"><b>Msc Computer Science. Human Practice Supervisor.</b> I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a0/Philipp_profile.jpg');"><br />
</div><br />
<br />
</div><br />
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<div class="gap"><br />
</div><br />
<br />
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<br />
<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/a/ae/Yanika_profile.jpg');"><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Yanika Borg</p> <br />
<p class="body_text"><b> PhD Student. Bacterial Lab Supervisor.</b> When I’m not working on my PhD in Synthetic Biology, I am supervising Spotless Mind’s bacterial team. My role is to oversee all experiments carried out on E. coli, to demonstrate molecular cloning techniques to the team, and to calm Andy down on a daily basis. This is my second year supervising iGEM at UCL, and I love the whole experience.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Alex Kinna</p> <br />
<p class="body_text"><b>PhD Student. Mammalian Lab Supervisor.</b> I am a 2nd year PhD student studying biochemical and protein engineering. My role is to advise and support mammalian cell culture, testing of circuits in mammalian cells and production of target proteins.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/6/6c/Kinna_profile.jpg');"><br />
</div><br />
<br />
</div><br />
<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="major_title">SCIENTIFIC ADVICE</p><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>In the course of the development of our idea, we consulted synthetic biologists, neuroscientists, neurosurgeons, psychiatrists and geneticists and took on board their feedback in order to develop our idea and add the detail to our genetic circuit. We show what advice we received here and how this advice was incorporated into our final project.</p></b><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Dr. Jeremy Cook</b> is a senior lecturer and the programme tutor for the Neuroscience Bsc at UCL. His research interests concern the development the visual system, including the embryonic emergence of retinal cell patterns. He advised us to carefully consider the neurosurgical implications of our project, noting the preferability of an autograph of microglia, and the need to design our circuit so that the microglia only become de-activated at plaques, because a degree of activation is required for chemotaxis. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Dr. John Scholes</b> is an honorary senior lecturer, and lectures on the Neuroscience Bsc course at UCL. He supported the idea of using BDNF in the circuit in order to stop cell cycle re-entry in AD and suggested ApoE as a possible circuit component, as it could increase the activity of our chosen protease. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Dr. Jennifer Pococks'</b> research involves cell signaling in neurodegenerative dieseases and this onvolves the study of microglia in the context of AD. She advised the team on using microglia in the lab.<br />
</div><br />
</div><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>Professor Patrick Haggard</b> is a prominent figure in neuroethical debate, Patrick Haggard is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, UCL. We are thankful to him for providing inspiration and being a sounding board for our neuroethical investigations, and for agreeing to be filmed as part of our documentary. <br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor John Powell</b> is a geneticist in the Department of Neuroscience and Psychological Medicine at Kings College London. His research interests are in the application of human genetics to the study of neurological and psychiatric disorders; in schizophrenia and autism. He helped direct our theoretical work on how synthetic neurobiology could be expanded to different brain conditions, therapies and enhancements.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor Stephen Hart</b> works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We are thankful for him on his advice concerning how to transfect native microglia in vivo. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using lipid-peptide nanocomplexes. This result of his was un-expected as his team had been trying to transfect cancerous cells in rat brains but increases the feasibility of our idea.<br />
<br />
<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>content</b><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="major_title">OTHER ACKNOWLEDGMENTS</p><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>content</b></p><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>content</b><br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><p class="body_text"><b>Lubmilla Ruban</b> is a stem cell biologist in UCL's Biochemical Engineering Department, who manages the Cell Culture, Cell Bioprocessing and the Liquid Nitrogen facilities. We are thankful to her for teaching us the basics of mammalian cell culture, including how to passage cells and how to use the essential equipment of the mammalian labs. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Sean Tuite</b> is a first year undergraduate film student to whom we owe thanks for his help in creating our documentary as cameraman and editor.<br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
</div><br />
<br />
</div><br />
<br />
<script type="text/javascript" src="https://2013.igem.org/Team:UCL/static/footer.js?action=raw&ctype=text/javascript"> <br />
</script><br />
<br />
</body><br />
</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T02:48:49Z<p>AlexBates: </p>
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<div class="gap"><br />
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<p class="major_title">SUPERVISORS</p><br />
<div class="gap"><br />
</div><br />
<div class="row_small"><br />
<br />
<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/1/10/Darren_profile.jpg');"><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Dr. Darren Nesbeth</p> <br />
<p class="body_text"><b>Lecturer in Synthetic and Molecular Biology.</b> Supervisor and overall co-ordinator of iGEM at UCL. Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Philipp Boeing</p> <br />
<p class="body_text"><b>Msc Computer Science. Human Practice Supervisor.</b> I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a0/Philipp_profile.jpg');"><br />
</div><br />
<br />
</div><br />
<br />
<div class="gap"><br />
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<br />
<div class="row_small"><br />
<br />
<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/a/ae/Yanika_profile.jpg');"><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Yanika Borg</p> <br />
<p class="body_text"><b> PhD Student. Bacterial Lab Supervisor.</b> When I’m not working on my PhD in Synthetic Biology, I am supervising Spotless Mind’s bacterial team. My role is to oversee all experiments carried out on E. coli, to demonstrate molecular cloning techniques to the team, and to calm Andy down on a daily basis. This is my second year supervising iGEM at UCL, and I love the whole experience.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Alex Kinna</p> <br />
<p class="body_text"><b>PhD Student. Mammalian Lab Supervisor.</b> I am a 2nd year PhD student studying biochemical and protein engineering. My role is to advise and support mammalian cell culture, testing of circuits in mammalian cells and production of target proteins.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/6/6c/Kinna_profile.jpg');"><br />
</div><br />
<br />
</div><br />
<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="major_title">SCIENTIFIC ADVICE</p><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>In the course of the development of our idea, we consulted synthetic biologists, neuroscientists, neurosurgeons, psychiatrists and geneticists and took on board their feedback in order to develop our idea and add the detail to our genetic circuit. We show what advice we received here and how this advice was incorporated into our final project.</p></b><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Dr. Jeremy Cook</b> is a senior lecturer and the programme tutor for the Neuroscience Bsc at UCL. His research interests concern the development the visual system, including the embryonic emergence of retinal cell patterns. He advised us to carefully consider the neurosurgical implications of our project, noting the preferability of an autograph of microglia, and the need to design our circuit in such a way that the microglia only become de-activated at plaques, because a degree of activation is required for chemotaxis. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Dr. John Scholes</b> is an honorary senior lecturer, and lectures on the Neuroscience Bsc course at UCL. He supported the idea of using BDNF in the circuit in order to stop cell cycle re-entry in AD and suggested ApoE as a possible circuit component, as it could increase the activity of our chosen protease. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Dr. Jennifer Pococks'</b> research involves cell signaling in neurodegenerative dieseases and this onvolves the study of microglia in the context of AD. She advised the team on using microglia in the lab.<br />
</div><br />
</div><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>Professor Patrick Haggard</b> is a prominent figure in neuroethical debate, Patrick Haggard is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, UCL. We are thankful to him for providing inspiration and being a sounding board for our neuroethical investigations, and for agreeing to be filmed as part of our documentary. <br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor John Powell</b> is a geneticist in the Department of Neuroscience and Psychological Medicine at Kings College London. His research interests are in the application of human genetics to the study of neurological and psychiatric disorders; in schizophrenia and autism. He helped direct our theoretical work on how synthetic neurobiology could be expanded to different brain conditions, therapies and enhancements.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Professor Stephen Hart</b> works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We are thankful for him on his advice concerning how to transfect native microglia in vivo. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using lipid-peptide nanocomplexes. Interestingly, this result of his was un-expected as his team had been trying to transfect cancerous cells in rat brains but increases the feasibility of our idea.<br />
<br />
<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>content</b><br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<br />
<div class="gap"><br />
</div><br />
<div class="gap"><br />
</div><br />
<p class="major_title">OTHER ACKNOWLEDGMENTS</p><br />
<div class="gap"><br />
</div><br />
<br />
<div class="row_small"><br />
<br />
<div class="col_1"><p class="body_text"><b>content</b></p><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>content</b><br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><p class="body_text"><b>Lubmilla Ruban</b> is a stem cell biologist in UCL's Biochemical Engineering Department, who manages the Cell Culture, Cell Bioprocessing and the Liquid Nitrogen facilities. We are thankful to her for teaching us the basics of mammalian cell culture, including how to passage cells and how to use the essential equipment of the mammalian labs. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Sean Tuite</b> is a first year undergraduate film student to whom we owe thanks for his help in creating our documentary as cameraman and editor.<br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
</div><br />
<br />
</div><br />
<br />
<script type="text/javascript" src="https://2013.igem.org/Team:UCL/static/footer.js?action=raw&ctype=text/javascript"> <br />
</script><br />
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</body><br />
</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T02:47:52Z<p>AlexBates: </p>
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<!-- START CONTENT ----------------------------------------------------------------------------------------------------><br />
<div class="gap"><br />
</div><br />
<p class="major_title">SUPERVISORS</p><br />
<div class="gap"><br />
</div><br />
<div class="row_small"><br />
<br />
<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/1/10/Darren_profile.jpg');"><br />
</div><br />
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<div class="col_1"><br />
<br />
<p class="minor_title">Dr. Darren Nesbeth</p> <br />
<p class="body_text"><b>Lecturer in Synthetic and Molecular Biology.</b> Supervisor and overall co-ordinator of iGEM at UCL. Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning.</p><br />
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<p class="minor_title">Philipp Boeing</p> <br />
<p class="body_text"><b>Msc Computer Science. Human Practice Supervisor.</b> I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!<br />
</p><br />
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<p class="minor_title">Yanika Borg</p> <br />
<p class="body_text"><b> PhD Student. Bacterial Lab Supervisor.</b> When I’m not working on my PhD in Synthetic Biology, I am supervising Spotless Mind’s bacterial team. My role is to oversee all experiments carried out on E. coli, to demonstrate molecular cloning techniques to the team, and to calm Andy down on a daily basis. This is my second year supervising iGEM at UCL, and I love the whole experience.</p><br />
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<p class="minor_title">Alex Kinna</p> <br />
<p class="body_text"><b>PhD Student. Mammalian Lab Supervisor.</b> I am a 2nd year PhD student studying biochemical and protein engineering. My role is to advise and support mammalian cell culture, testing of circuits in mammalian cells and production of target proteins.<br />
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<p class="major_title">SCIENTIFIC ADVICE</p><br />
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<div class="col_1"><p class="body_text"><b>In the course of the development of our idea, we consulted synthetic biologists, neuroscientists, neurosurgeons, psychiatrists and geneticists and took on board their feedback in order to develop our idea and add the detail to our genetic circuit. We show what advice we received here and how this advice was incorporated into our final project.</p></b><br />
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<p class="body_text"><b>Dr. Jeremy Cook</b> is a senior lecturer and the programme tutor for the Neuroscience Bsc at UCL. His research interests concern the development the visual system, including the embryonic emergence of retinal cell patterns. He advised us to carefully consider the neurosurgical implications of our project, noting the preferability of an autograph of microglia, and the need to design our circuit in such a way that the microglia only become de-activated at plaques, because a degree of activation is required for chemotaxis. <br />
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<p class="body_text"><b>Dr. John Scholes</b> is an honorary senior lecturer, and lectures on the Neuroscience Bsc course at UCL. He supported the idea of using BDNF in the circuit in order to stop cell cycle re-entry in AD and suggested ApoE as a possible circuit component, as it could increase the activity of our chosen protease. <br />
</p><br />
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</div><br />
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<div class="col_2"><p class="body_text"><b>Dr. Jennifer Pococks'</b> research involves cell signaling in neurodegenerative dieseases and this onvolves the study of microglia in the context of AD. She advised the team on using microglia in the lab.<br />
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<div class="col_1"><p class="body_text"><b>Professor Patrick Haggard</b> is a prominent figure in neuroethical debate, Patrick Haggard is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, UCL. We are thankful to him for providing inspiration and being a sounding board for our neuroethical investigations, and for agreeing to be filmed as part of our documentary. <br />
</div><br />
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<div class="col_1"><br />
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<p class="body_text"><b>Professor John Powell</b> is a geneticist in the Department of Neuroscience and Psychological Medicine at Kings College London. His research interests are in the application of human genetics to the study of neurological and psychiatric disorders; in schizophrenia and autism. He helped direct our theoretical work on how synthetic neurobiology could be expanded to different brain conditions, therapies and enhancements.<br />
</p><br />
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<br />
<p class="body_text"><b>Professor Stephen Hart</b> works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We are thankful for him on his advice concerning how to transfect native microglia in vivo. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using lipid-peptide nanocomplexes. Interestingly, this result of his was un-expected as his team had been trying to transfect cancerous cells in rat brains. Their unintended discovery is a great boon for our idea.<br />
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<div class="col_2"><p class="body_text"><b>content</b><br />
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<p class="major_title">OTHER ACKNOWLEDGMENTS</p><br />
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<div class="col_1"><p class="body_text"><b>content</b></p><br />
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<p class="body_text"><b>content</b><br />
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<p class="body_text"><p class="body_text"><b>Lubmilla Ruban</b> is a stem cell biologist in UCL's Biochemical Engineering Department, who manages the Cell Culture, Cell Bioprocessing and the Liquid Nitrogen facilities. We are thankful to her for teaching us the basics of mammalian cell culture, including how to passage cells and how to use the essential equipment of the mammalian labs. <br />
</p><br />
<br />
</div><br />
<br />
<div class="col_2"><p class="body_text"><b>Sean Tuite</b> is a first year undergraduate film student to whom we owe thanks for his help in creating our documentary as cameraman and editor.<br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<br />
<p class="major_title">August</p><br />
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<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">September</p><br />
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<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
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<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<p class="body_text"><br />
Narration filming for documentary continued in the UCL campus. <br />
</p><br />
<p class="body_text"><br />
Apart from that, Professor John Powell was very kind to accept to be interviewed by our team.<br />
</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<p class="body_text"><br />
Alex finished the bioinformatics work at Cancer Research UK in the BMM lab. The programme intended to be run with cancer data to produced results for a paper being written in the lab. Alex begins to run the programme with Alzheimer's data to see how our circuit could be improved.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<p class="body_text"><br />
The Narration for our documentary was continued with the interview of Professor Patrick Haggard followed by interviewing Professor Stephen Hart. <br />
</p> <br />
<p class="body_text"><br />
More information on Documentary subsection of Human Practice section.<br />
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<p class="minor_title">Dr. Darren Nesbeth</p> <br />
<p class="body_text"><b>Lecturer in Synthetic and Molecular Biology.</b> Supervisor and overall co-ordinator of iGEM at UCL. Lecturer in Synthetic Biology at the Department of Biochemical Engineering, who has been responsible for overseeing the iGEM competition at UCL for many years! Loves to eat porridge and watch vintage VHS films when away from iGEM planning.</p><br />
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<p class="minor_title">Philipp Boeing</p> <br />
<p class="body_text"><b>Msc Computer Science. Human Practice Supervisor.</b> I have been leading iGEM teams at UCL since 2011, including last year’s Plastic Republic team. This year, I principally supervise team Spotless Mind on Human Practice, as well as general iGEM best practice. Apart from iGEM, I spend my time on SynBioSoc and DIYbio. Diversity!<br />
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<p class="minor_title">Yanika Borg</p> <br />
<p class="body_text"><b> PhD Student. Bacterial Lab Supervisor.</b> When I’m not working on my PhD in Synthetic Biology, I am supervising Spotless Mind’s bacterial team. My role is to oversee all experiments carried out on E. coli, to demonstrate molecular cloning techniques to the team, and to calm Andy down on a daily basis. This is my second year supervising iGEM at UCL, and I love the whole experience.</p><br />
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<p class="minor_title">Alex Kinna</p> <br />
<p class="body_text"><b>PhD Student. Mammalian Lab Supervisor.</b> I am a 2nd year PhD student studying biochemical and protein engineering. My role is to advise and support mammalian cell culture, testing of circuits in mammalian cells and production of target proteins.<br />
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<p class="major_title">SCIENTIFIC ADVICE</p><br />
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<div class="col_1"><p class="body_text"><b>In the course of the development of our idea, we consulted synthetic biologists, neuroscientists, neurosurgeons, psychiatrists and geneticists and took on board their feedback in order to develop our idea and add the detail to our genetic circuit. We show what advice we received here and how this advice was incorporated into our final project.</p></b><br />
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<p class="body_text"><b>Dr. Jeremy Cook</b> is a senior lecturer and the programme tutor for the Neuroscience Bsc at UCL. His research interests concern the development the visual system, including the embryonic emergence of retinal cell patterns. He advised us to carefully consider the neurosurgical implications of our project, noting the preferability of an autograph of microglia, and the need to design our circuit in such a way that the microglia only become de-activated at plaques, because a degree of activation is required for chemotaxis. <br />
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<p class="body_text"><b>Dr. John Scholes</b> is an honorary senior lecturer, and lectures on the Neuroscience Bsc course at UCL. He supported the idea of using BDNF in the circuit in order to stop cell cycle re-entry in AD and suggested ApoE as a possible circuit component, as it could increase the activity of our chosen protease. <br />
</p><br />
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<div class="col_2"><p class="body_text"><b>Dr. Jennifer Pococks'</b> research involves cell signaling in neurodegenerative dieseases and this onvolves the study of microglia in the context of AD. She advised the team on using microglia in the lab.<br />
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<p class="body_text"><b>content</b><br />
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<p class="body_text"><b>content</b> <br />
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<p class="major_title">OTHER ACKNOWLEDGMENTS</p><br />
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<p class="body_text"><p class="body_text"><b>Lubmilla Ruban</b> is a stem cell biologist in UCL's Biochemical Engineering Department, who manages the Cell Culture, Cell Bioprocessing and the Liquid Nitrogen facilities. We are thankful to her for teaching us the basics of mammalian cell culture, including how to passage cells and how to use the essential equipment of the mammalian labs. <br />
</p><br />
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<div class="col_2"><p class="body_text"><b>Sean Tuite</b> is a first year undergraduate film student to whom we owe thanks for his help in creating our documentary as cameraman and editor.<br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">October</p><br />
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<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
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<p class="major_title">October</p><br />
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<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">MEDAL CRITERIA</p><br />
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<p class="minor_title">Bronze Medal</p><br />
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• We have registered our <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">team</a> of eleven undergraduates. <br />
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• We have completed the judging form.<br />
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• We have created a beautiful team wiki in keeping with the iGEM guidelines.<br />
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• We have a poster and presentation ready for the regional jamboree in Lyon.<br />
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• We have developed two new <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">BioBricks</a> and have submitted them to the iGEM Registry<br />
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<p class="minor_title">Silver Medal</p><br />
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• We have proved our zeocin resistance BioBrick <a href="https://2013.igem.org/Team:UCL/Project/Parts" target="_blank">worked as expected</a> by characterising it in HeLa cells. We did this by by creating a zeocin kill curve with and without our <a href="https://2013.igem.org/Team:UCL/Project/Marker" target="_blank">zeocin resistance</a> BioBrick. <br />
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• Due to the gravity of Alzheimer's disease and the perceived sovereignty of the brain, we have taken the ethics of using synthetic biological treatments very seriously. We produced a <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">neuroethics</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility</a> report, consulted numerous experts and provided a concise but detailed <a href="https://2013.igem.org/Team:UCL/Background" target="_blank">background</a> to our project, which shows how our proposed <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">genetic circuit</a> is advised by multiple theories for the causation of Alzheimer's pathology.<br />
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Gold Medal<br />
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• We collaborated with iGEM Westminster, by modelling how well their bed-bug killing device will operate in a bedroom as well advising them on how to run a speed debate using our format. One of our team members, Alex Bates, also attended as guest speaker at their speed debate event.<br />
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Full details on collaboration with iGEM Westminster can be found below.<br />
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• Our <a href="https://2013.igem.org/Team:UCL/Project/Circuit" target="_blank">Human Practices</a> deal with an entirely new area for iGEM and, indeed, almost a completely new avenue of research for synthetic biology as a field - the <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">fusion</a> of neuroscience and synthetic biology. We use a variety of strong methods for social and ethical analysis, and outreach. Please see the section below for a summary of what we achieved.<br />
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• Outside of this theme, we also engaged in outreach by training and advising the UCL Academy iGEM high school team. This was the first time a British iGEM team has helped run an iGEM HS team.<br />
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<p class="major_title">ABOVE AND BEYOND</p><br />
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<p class="minor_title">Project</p><br />
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• Developed and experimentally validated the first selectable marker (Zeocin) in a mammalian system (HeLa cells). <br />
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• Plan to use genetically engineered microglia cells; the first human brain cells to be used in iGEM, when they arrive so results will be available post jamboree. <br />
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<p class="minor_title">Human Practices</p><br />
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• We have looked at an entirely new ethical area for iGEM that has also essentially not been covered in academia; the neuroethics of genetic engineering. We have dubbed this <a href="https://2013.igem.org/Team:UCL/Practice" target="_blank">'Neuro-genethics'</a>. <br />
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• We have produced an extensive 20 page <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics" target="_blank">report</a> that looks into neuro-genethics and what synthetic biology could achieve in neuroscience.<br />
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• We have engaged the public on this topic by getting their opinions at the <a href="http://www.artscatalyst.org/" target="_blank">Arts Catalyst</a>, running a <a href="https://2013.igem.org/Team:UCL/Practice/Debate" target="_blank">speed debate</a> and a <a href="https://2013.igem.org/Team:UCL/Practice/TED" target="_blank">TED debate</a>, conducting an <a href="https://2013.igem.org/Team:UCL/Practice/Survey" target="_blank">online survey</a> and producing a <a href="https://2013.igem.org/Team:UCL/Practice/Documentary" target="_blank">documentary </a> on synthetic neurobiology.<br />
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• We ran one of iGEM's first <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank">creative writing competitions</a>, to gauge public opinion on brain modification and highligh5t the impact of fiction on society's views.<br />
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• Petcha Kutcha style presentation to enguage prospective students of the UCL Engineering Department about iGEM.<br />
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• We created an original memory bank, <a href="https://2013.igem.org/Team:UCL/Memories" target="_blank">Eternal Sunshine</a>, which highlights how precious memories are, indicating the desperate need to cure Alzheimer's disease. <br />
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• We created a <a href="https://2013.igem.org/Team:UCL/Practice/Report" target="_blank">feasibility report</a> on implementing our treatment.<br />
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• Attended and aided in the running of YSB 1.0 (held at UCL) along with other UK iGEM teams to discuss projects and potential collaborations. <br />
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• Through Eternal Sunshine and the creative writing competition we have successfully reached out to a diverse range of people from every corner of the globe, from the USA , to China, to Saudi Arabia and so on. <br />
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<p class="minor_title">Modelling</p><br />
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• We are the first iGEM team to use a <a href="https://2013.igem.org/Team:UCL/Modeling" target="_blank">protein network analysis approach</a>. Network based bioinformatics can feedback into synthetic biology by informing the choice of parts in therapeutic genetic circuits.<br />
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<p class="minor_title">Wiki</p><br />
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• We have developed an, original attractive wiki using art work by our artists in residence, <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Fong Yi Khoo</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Oran Maguire</a>.<br />
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• We have included an extensive neuroscience background section, which explains and compares multiple theories for the causation of Alzheimer's disease, so that readers can fully understand the pros and cons of our genetic circuit. <br />
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• We have included a full complement of citations that link to PubMed pages so that it is easy to see from where our ideas and explanations have been drawn, and which papers have inspired us.<br />
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<p class="minor_title">Collaboration with Westminster iGEM</p><br />
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<b>Model (ROBIN WRITES HERE)</b><br />
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Movement of bed-bugs towards blood meal<br />
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<p class="minor_title">Mentoring iGEM HS</p><br />
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Two members of the UCL iGEM team volunteered as advisers to the UCL Academy iGEM team – <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Ruxi</a> and <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Khaicheng</a>, under the guidance of Aurelija Grigonyte, a member of the UCL iGEM 2012 team. During the high school team’s brainstorming process, we provided them with guidance and resources for their research. We also supervised their lab work in the UCL Biochemical Engineering department.<br />
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UCL is the first university in the UK to be the sole sponsor of an academy – a non-selective mixed state school in our home borough of Camden. UCL Academy represents a unique opportunity to blur the boundaries between secondary and higher education.<br />
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The academy is one of the first UK high schools to participate in iGEM this year, and is the only UK team so far to have attended the <a href="https://2013hs.igem.org/Main_Page" target="_blank">High School iGEM Jamboree</a> at MIT, Boston. The team aimed to revolutionise the recycling industry by proposing a home system that converts cellulose into glucose, allowing the up-cycling of paper into a commercial product of bioplastic - polyhydroxybutyrate (PHB) <br />
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For information about their iGEM project, check out their wiki <a href="https://2013hs.igem.org/Team:UCL_Academy" target="_blank">here</a>. <br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Notebook/OctoberTeam:UCL/Notebook/October2013-10-05T02:24:38Z<p>AlexBates: </p>
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<p class="major_title">October</p><br />
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<p class="minor_title">1st October</p><br />
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Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
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<p class="minor_title">2nd October</p><br />
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The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
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<p class="minor_title">3rd October</p><br />
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Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
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FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
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Today we received the T-Shirts. <br />
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The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
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We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
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<p class="major_title">January</p><br />
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After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
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Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
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Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
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• Weight control yoghurt<br />
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• Anti-cancer yoghurt<br />
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• Zebrafish water cleaning system for Third World<br />
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• Athletic Drug testing<br />
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• Clean Urban Air<br />
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• Neural network with glowing bacteria and fibre optics<br />
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DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
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<p class="major_title">March</p><br />
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Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
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• Anti-cancer yoghurt<br />
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• Zebrafish<br />
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• Alzheimer's disease<br />
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• Neural Network<br />
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Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
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<p class="major_title">April & May</p><br />
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Exam period - iGEM work to commence full time after the slog through exams.<br />
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<p class="major_title">June</p><br />
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<p class="minor_title">5th June</p><br />
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Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
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Cancer research roles:<br />
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1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
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2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
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3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
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Considerations:<br />
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a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
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4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
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5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
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6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
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<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<p class="body_text"><br />
Narration filming for documentary continued in the UCL campus. <br />
</p><br />
<p class="body_text"><br />
Apart from that, Professor John Powell was very kind to accept to be interviewed by our team.<br />
</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<p class="body_text"><br />
Alex finished the bioinformatics work at Cancer Research UK in the BMM lab. The programme intended to be run with cancer data to produced results for a paper being written in the lab. Alex begins to run the programme with Alzheimer's data to see how our circuit could be improved.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<p class="body_text"><br />
The Narration for our documentary was continued with the interview of Professor Patrick Haggard followed by interviewing Professor Stephen Hart. <br />
</p> <br />
<p class="body_text"><br />
More information on Documentary subsection of Human Practice section.<br />
</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T02:12:02Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<div class="full_page"><br />
<br />
<div class="main_image"></div><br />
<br />
<p class="major_title">January</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">February</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
</div><br />
<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<p class="body_text"><br />
Narration filming for documentary continued in the UCL campus. <br />
</p><br />
<p class="body_text"><br />
Apart from that, Professor John Powell was very kind to accept to be interviewed by our team.<br />
</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<p class="body_text"><br />
The Narration for our documentary was continued with the interview of Professor Patrick Haggard followed by interviewing Professor Stephen Hart. <br />
</p> <br />
<p class="body_text"><br />
More information on Documentary subsection of Human Practice section.<br />
</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T02:07:00Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<div class="full_page"><br />
<br />
<div class="main_image"></div><br />
<br />
<p class="major_title">January</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">February</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
</div><br />
<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<p class="body_text"><br />
Narration filming for documentary continued in the UCL campus. <br />
</p><br />
<p class="body_text"><br />
Apart from that, Professor John Powell was very kind to accept to be interviewed by our team.<br />
</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T02:05:20Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<div class="full_page"><br />
<br />
<div class="main_image"></div><br />
<br />
<p class="major_title">January</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">February</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
</div><br />
<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<p class="body_text"><br />
Narration filming for documentary continued in the UCL campus. Apart from that, Professor John Powell was very kind to accept to be interviewed by our team.<br />
</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T02:01:11Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<div class="full_page"><br />
<br />
<div class="main_image"></div><br />
<br />
<p class="major_title">January</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">February</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
</div><br />
<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<p class="body_text"><br />
Bacterial Lab is experiencing some sparks of success. Possibly the ligated zeocin biobrick was achieved!<br />
</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<p class="body_text"><br />
Began the narration filming for the documentary. This start happened in the Grant Museum of Zooloy.<br />
</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<br />
</div><br />
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<p class="major_title">October</p><br />
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<div class="gap"><br />
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<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Practice/SurveyTeam:UCL/Practice/Survey2013-10-05T01:53:48Z<p>AlexBates: </p>
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<p class="major_title">Online Survey</p><br />
<p class="minor_title">Consulting Public Opinion</p><br />
<p class="body_text"><br />
The online survey serves the purpose of exploring the public opinion on synthetic biology and neuro-engineering. We wanted to research who are the ones that are interested and concerned about this line of research, and what their opinion are on different forms of neuro-genetic engineering, how much alteration is deemed appropriate, and what are the major concerns of neuro-genetic engineering. <br />
</p><br />
<br />
<br />
<br />
<p class="body_text"><b> <br />
How comfortable are you about using gene products from genetically modified (GM) bacteria to benefit human health? These products are created outside of the body, and administered as drugs.<br />
</b><br />
</p><br />
<br />
<br />
<p class="body_text"><br />
This concludes that the majority (53.1%) of the people who responded to the survey are positive about using gene products from genetically modified bacteria created outside of the body through drug delivery to benefit human health. This indicates a positive respond from the public in that they are comfortable using such technology for the benefit of human kind. <br />
</p><br />
<br />
<br />
<br />
<p class="body_text"><b> <br />
How comfortable are you about using gene products from GM animals to benefit human health? These products are taken from animals, and may involve killing the animal.<br />
</b>['</p><br />
<br />
<br />
<br />
<p class="body_text"><br />
The results from this question is relatively mixed, this indicates roughly that the public is generally not in favour of using gene products from GM animals to benefit human health, these products are taken from animals and may involve killing the animal. A big portion (34.4%) is indifferent about the matter and (28.1%) is in favour of using gene products from GM animals. This indicates that the ethical side of using animals for drug making is justified if it is seen to benefit the human kind. <br />
</p><br />
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<br />
<br />
<p class="body_text"><b> <br />
How comfortable do you feel about genetically modified organisms (GMOs) being inserted into your body (excluding the nervous system) for health benefits? For example, ingesting a yoghurt with live GM bacteria or inserting GM human cells to tackle cancer?<br />
</b><br />
</p><br />
<br />
<br />
<br />
<p class="body_text"><br />
This diagram indicates that the majority (59.4%) are in favour of both the use of GM human cells in the body (excluding the nervous system) for health benefits such as ingesting a yoghurt with live GM bacteria or inserting GM human cells to tackle cancer. Second in line is people in favour of inserting live GM bacteria into the body through ingestion to tackle cancer cells, but not genetically modifying native human cells to tackle cancer. This data concludes that the public is generally more comfortable to have foreign substance be injected into the body rather than having their own cells modified. In general, the responses show that people are more in favour of using genetically modification to treat health diseases. <br />
</p><br />
<br />
<br />
<br />
<p class="body_text"><b> <br />
Would you feel comfortable with a medical procedure that alters gene expression in the brain?<br />
</b><br />
</p><br />
<br />
<p class="body_text"><br />
This question concludes that most people are in favour of altering brain cells as a medical procedure against dementia, though there is only a slight difference between those who are against and those who are in favour. <br />
</p><br />
<br />
<p class="body_text"><b> <br />
Would you support the use of genetic neuro-enhancements in society, which alter gene expression / connectivity in the brain? These procedures could alter intellectual and cognitive traits, from intelligence to memory.<br />
</b><br />
</p><br />
<br />
<p class="body_text"><br />
Data analysis indicates that the majority is against large enhancements involving alteration of intellectual and cognitive traits from intelligence to memory,as this concerns a relative sensitive topic concerning identity modification. It is interesting to see how much alteration of the brain the public deem to be appropriate <br />
</p><br />
<br />
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<br />
<br />
<p class="body_text"><b> <br />
Would you generally support the use of genetic neuro-therapy in society? This could reduce, for example, violent behaviour in criminals, sexual preferences of paedophiles, etc., as well as introduce 'cosmetic psychology', the ability to choose religiosity, optimism/pessimism, sleeping patterns, sexual orientation, etc?<br />
</b><br />
</p><br />
<br />
<p class="body_text"><br />
The majority is against genetic neuro-therapy for cosmetic psychology purposes, however using genetic neuro-therapy for rehabilitation to reduce criminality is somehow justified. <br />
</p><br />
<br />
<p class="body_text"><b> <br />
What do you feel are the main driving influences behind your answers to the last three questions concerning gene manipulation in brain cells?<br />
</b><br />
</p><br />
<br />
<br />
<p class="body_text"><br />
The last question concludes that the public opinion on the driving influences behind gene manipulation in the brain comes largely from the benefits/ costs to society, the next one comes from benefits/ costs to the happiness of the treatment user. Religious moral view take up a very little percentage of the reply. It appears that more people care largely about the benefit and cost to society rather than the benefit and costs to the health of the treatment user.<br />
</p><br />
<br />
<p class="body_text"> <br />
In conclusion, the result seemed to give a nice overview of the public opinion of synthetic biology and neuro-genetic engineering. Data analysis and a graphic display of the results conclude that most of the young people care about synthetic biology more than the older generation. The greatest split of opinion is still the debate between whether genetic engineering of native cells in the brain is acceptable or not. This indicates that as a relatively new field of therapy, the public is still uncertain whether the treatment is safe and effective to use. The cost to society is also a concern as it appears to be relatively expensive to research and use genetically engineer cells as a medical treatment. <br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T01:53:28Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
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<p class="major_title">January</p><br />
<div class="full_row"><br />
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<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
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<br />
<br />
<p class="major_title">February</p><br />
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<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
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<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
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<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
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<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
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<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
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<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh. Obtained new stocks of valuable pSB1C3.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/NotebookTeam:UCL/Notebook2013-10-05T01:51:57Z<p>AlexBates: </p>
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<p class="body_text"><a href="https://2013.igem.org/Team:UCL/Notebook/January">January</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/February">February</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/March">March</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/April">April</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/May">May</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/June">June</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/July">July</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/August">August</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/September">September</a> | <a href="https://2013.igem.org/Team:UCL/Notebook/October">October</a> <br />
</p> <br />
</div><br />
<br />
<div class="full_page"><br />
<br />
<div class="main_image"></div><br />
<br />
<p class="major_title">January</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project. <br />
</p><br />
<p class="body_text"><br />
Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">February</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:<br />
</p><br />
<p class="body_text"><br />
• Weight control yoghurt<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish water cleaning system for Third World<br />
</p><br />
<p class="body_text"><br />
• Athletic Drug testing<br />
</p><br />
<p class="body_text"><br />
• Clean Urban Air<br />
</p><br />
<p class="body_text"><br />
• Neural network with glowing bacteria and fibre optics<br />
</p><br />
<p class="body_text"><br />
DIY SynBio group at <a href="http://www.artscatalyst.org" target="_blank">The Arts Catalyst</a> were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.<br />
</p><br />
</div><br />
<br />
<br />
<p class="major_title">March</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:<br />
</p><br />
<p class="body_text"><br />
• Anti-cancer yoghurt<br />
</p><br />
<p class="body_text"><br />
• Zebrafish<br />
</p><br />
<p class="body_text"><br />
• Alzheimer's disease<br />
</p><br />
<p class="body_text"><br />
• Neural Network<br />
</p><br />
</p><br />
<p class="body_text"><br />
Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.<br />
</p><br />
</div><br />
<br />
<p class="major_title">April & May</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
Exam period - iGEM work to commence full time after the slog through exams.<br />
</p><br />
</div><br />
<br />
<p class="major_title">June</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">5th June</p><br />
<p class="body_text"><br />
Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:<br />
</p><br />
<p class="body_text"><br />
Cancer research roles:<br />
</p><br />
<p class="body_text"><br />
1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…<br />
</p><br />
<p class="body_text"><br />
2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.<br />
</p><br />
<p class="body_text"><br />
3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.<br />
</p><br />
<p class="body_text"><br />
Considerations:<br />
</p><br />
<p class="body_text"><br />
a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? <br />
b. If it is a toxin, what sort of biosynthetic pathway is required?<br />
c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin?<br />
d. Are there any treatments which we can take advantage of specifically because we are using bacteria? <br />
e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?<br />
</p><br />
<p class="body_text"><br />
4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?<br />
</p><br />
<p class="body_text"><br />
5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?<br />
</p><br />
<p class="body_text"><br />
6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.<br />
</p><br />
<br />
<p class="minor_title">7th June</p><br />
<p class="body_text"><br />
The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.<br />
</p><br />
<p class="minor_title">11th June</p><br />
<p class="body_text"><br />
looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.<br />
</p><br />
<p class="body_text"><br />
We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.<br />
</p><br />
<p class="body_text"><br />
From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:<br />
</p><br />
<p class="body_text"><br />
1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.<br />
</p><br />
<p class="body_text"><br />
2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.<br />
</p><br />
<p class="body_text"><br />
Goals for the end of this week: <br />
</p><br />
<p class="body_text"><br />
- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios<br />
</p><br />
<p class="body_text"><br />
-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.<br />
</p><br />
<p class="minor_title">12th June</p><br />
<p class="body_text"><br />
Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.<br />
</p><br />
<p class="body_text"><br />
Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks. <br />
</p><br />
<p class="body_text"><br />
We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.<br />
</p><br />
<p class="body_text"><br />
Andy searched potential cancer killer molecules:<br />
</p><br />
<p class="body_text"><br />
- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html)<br />
- Tumor Necrosis Factor, Histamine - induces inflammation<br />
- HAMLET (human a-lactalbumin) - induces apoptosis <br />
- endostatin, thrombospondin - reduce cancer growth<br />
</p><br />
<p class="body_text"><br />
Weiling looked at potential promotors: <br />
</p><br />
<p class="body_text"><br />
- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule<br />
- Lux pR promotor<br />
- Lld promoter<br />
- Vgb promotor <br />
- HIP-1<br />
</p><br />
<p class="body_text"><br />
(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96) <br />
</p><br />
<p class="body_text"><br />
For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very <br />
good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf<br />
</p><br />
<p class="body_text"><br />
We need to register this part!<br />
</p><br />
<br />
<p class="minor_title">13th June</p><br />
<p class="body_text"><br />
Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.<br />
</p><br />
<p class="body_text"><br />
Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.<br />
</p><br />
<p class="body_text"><br />
Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.<br />
</p><br />
<p class="body_text"><br />
Catrin - General project research<br />
</p><br />
<p class="body_text"><br />
Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.<br />
</p><br />
<p class="body_text"><br />
We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).<br />
</p><br />
<p class="body_text"><br />
Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:<br />
</p><br />
<p class="body_text"><br />
Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.<br />
</p><br />
<p class="body_text"><br />
Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.<br />
</p><br />
<p class="body_text"><br />
Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.<br />
</p><br />
<br />
<p class="minor_title">14th June</p><br />
<p class="body_text"><br />
Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages<br />
</p><br />
<p class="body_text"><br />
Robin - Coding in HTML for website<br />
</p><br />
<p class="body_text"><br />
Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.<br />
</p><br />
<p class="body_text"><br />
Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.<br />
</p><br />
<p class="minor_title">17th June</p><br />
<p class="body_text"><br />
The group had a meeting to discuss what had been achieved so far and what needed to be done today. <br />
</p><br />
<p class="body_text"><br />
Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.<br />
</p><br />
<p class="body_text"><br />
Robin - Continued on website coding.<br />
</p><br />
<p class="body_text"><br />
Weiling & Catrin - Researched for project sponsors and potential contacts.<br />
</p><br />
<p class="body_text"><br />
Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.<br />
</p><br />
<p class="minor_title">18th June</p><br />
<p class="body_text"><br />
The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.<br />
</p><br />
<p class="body_text"><br />
The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.<br />
</p><br />
<p class="body_text"><br />
The team also had a social gathering: pizza for lunch.<br />
</p><br />
<p class="minor_title">19th June</p><br />
<p class="body_text"><br />
The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.<br />
</p><br />
<p class="minor_title">20th June</p><br />
<p class="body_text"><br />
Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.<br />
</p><br />
<p class="body_text"><br />
The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.<br />
</p><br />
<p class="minor_title">21st June</p><br />
<p class="body_text"><br />
Tom - Continued wiki design, coding and content uploads.<br />
Alex - Continued to redraft the proposal for Alzheimer's<br />
StJohn - Continued to redraft the proposal for Cancer<br />
</p><br />
<p class="body_text"><br />
KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email<br />
</p><br />
<p class="body_text"><br />
The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.<br />
</p><br />
<p class="minor_title">24th June</p><br />
<p class="body_text"><br />
Tom continued wiki design whilst the rest of the group performed research.<br />
</p><br />
<p class="body_text"><br />
Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided. <br />
</p><br />
<p class="body_text"><br />
A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.<br />
</p><br />
<br />
<p class="minor_title">25th June</p><br />
<p class="body_text"><br />
The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.<br />
</p><br />
<br />
<p class="minor_title">27th June</p><br />
<p class="body_text"><br />
The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.<br />
</p><br />
<br />
<p class="minor_title">28th June</p><br />
<p class="body_text"><br />
Tom presented to prospective students about the iGEM project for the day.<br />
</p><br />
<p class="body_text"><br />
Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.<br />
</p><br />
<p class="body_text"><br />
KC, Robin and StJohn discussed lab protocols and also modelling ideas.<br />
</p><br />
<p class="minor_title">29th June</p><br />
<p class="body_text"><br />
Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.<br />
</p><br />
</div><br />
<br />
<p class="major_title">July</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st July</p><br />
<p class="body_text"><br />
Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.<br />
</p><br />
<p class="body_text"><br />
Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.<br />
</p><br />
<p class="body_text"><br />
StJohn & Ruxi – Formed project proposals for the laboratory experiments.<br />
</p><br />
<p class="minor_title">2nd July</p><br />
<p class="body_text"><br />
The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.<br />
</p><br />
<p class="minor_title">3rd July</p><br />
<p class="body_text"><br />
The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.<br />
</p><br />
<br />
<p class="minor_title">4th July</p><br />
<p class="body_text"><br />
The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.<br />
</p><br />
<p class="body_text"><br />
Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group. <br />
</p><br />
<br />
<p class="minor_title">5th July</p><br />
</p><br />
<p class="body_text"><br />
Tom & Catrin – Worked on the poster and finished it, as well as the presentation<br />
</p><br />
<p class="body_text"><br />
Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.<br />
</p><br />
<p class="body_text"><br />
KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.<br />
</p><br />
<p class="minor_title">8th July</p><br />
<p class="body_text"><br />
Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.<br />
</p><br />
<br />
<p class="minor_title">9th July</p><br />
<br />
<p class="body_text"><br />
Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.<br />
</p><br />
<br />
<p class="minor_title">10th July</p><br />
<p class="body_text"><br />
The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.<br />
</p><br />
<p class="minor_title">11th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">12th July</p><br />
<p class="body_text"><br />
The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.<br />
</p><br />
<p class="minor_title">13th July</p><br />
<p class="body_text"><br />
YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.<br />
</p><br />
<p class="minor_title">15th July</p><br />
<p class="body_text"><br />
First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.<br />
</p><br />
<p class="minor_title">16th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.<br />
</p><br />
<p class="body_text"><br />
KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.<br />
</p><br />
<p class="minor_title">17th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.<br />
</p><br />
<p class="body_text"><br />
KC & Alex – Started mammalian cell lab induction.<br />
</p><br />
<p class="body_text"><br />
The team then met with artists to further develop the branding of the whole project.<br />
</p><br />
<p class="minor_title">18th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<p class="minor_title">19th July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.<br />
</p><br />
<p class="body_text"><br />
StJohn & KC – Primer design for the PCR protocols<br />
</p><br />
<p class="body_text"><br />
Alex – Continued work on ethics and feasibility report & bioinformatics<br />
</p><br />
<br />
<p class="minor_title">22nd July</p><br />
<p class="body_text"><br />
Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.<br />
</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.<br />
</p><br />
<p class="minor_title">23rd July</p><br />
<p class="body_text"><br />
Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.<br />
</p><br />
<p class="body_text"><br />
StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.<br />
</p><br />
<p class="body_text"><br />
Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.<br />
</p><br />
<br />
<p class="minor_title">24th July</p><br />
<p class="body_text"><br />
Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.<br />
</p><br />
<p class="body_text"><br />
The entire team was sent the information regarding mammalian lab aseptic techniques.<br />
StJohn analised an <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277080/" target="_blank"> article</a> on Microglia function in Alzheimer’s disease.<br />
</p><br />
<p class="body_text"><br />
Alex gathered more <a href="http://www.scielo.br/pdf/bjmbr/v38n7/v38n7a03.pdf" target="_blank"> information</a> regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.<br />
</p><br />
<p class="body_text"><br />
The team decided to meet over for a barbeque on the 7th of August.<br />
</p><br />
<br />
<p class="minor_title">25th July</p><br />
<p class="body_text"><br />
Oran came to the lab and was introduced to the lab routine and to the activities on going.<br />
The team met again in the Student Anatomy hub to continue research on useful articles.<br />
</p><br />
<br />
<p class="minor_title">26th July</p><br />
<p class="body_text"><br />
A summary of the week lab work:<br />
</p><br />
<p class="body_text"><br />
- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.<br />
</p><br />
<p class="body_text"><br />
- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).<br />
</p><br />
<p class="body_text"><br />
- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.<br />
</p><br />
<p class="body_text"><br />
- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.<br />
</p><br />
<p class="body_text"><br />
The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.<br />
</p><br />
<br />
<p class="minor_title">29th July</p><br />
<p class="body_text"><br />
An important day for our team! The project name “Spotless mind” was chosen!<br />
</p><br />
<p class="body_text"><br />
The MathWorks license for the 2013 iGEM student competition has been created.<br />
</p><br />
<p class="body_text"><br />
The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.<br />
</p><br />
<br />
<p class="minor_title">30th July</p><br />
<p class="body_text"><br />
The entire team is involved in organising the speed debate taking place tomorrow, 31st.<br />
FYi and Oran produced a nice poster. <a href="https://scontent-b.xx.fbcdn.net/hphotos-prn1/q71/s720x720/1098138_10151827937531617_373872629_n.jpg" target="_blank"> debate poster</a> and a new logo!<br />
</p><br />
<br />
<p class="minor_title">31st July</p><br />
<p class="body_text"><br />
We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.<br />
</p><br />
<p class="body_text"><br />
The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm<br />
</p><br />
</div><br />
<p class="major_title">August</p><br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="minor_title">1st August</p><br />
<p class="body_text"><br />
Bacterial lab had good results today in the preparation of a new stock of competent cells.<br />
In the evening we celebrated the success of the speed debate.<br />
</p><br />
<p class="minor_title">2nd August</p><br />
<p class="body_text"><br />
Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter.<br />
The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.<br />
</p><br />
<p class="body_text"><br />
We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.<br />
</p><br />
<p class="body_text"><br />
Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.<br />
</p><br />
<br />
<p class="minor_title">5th August</p><br />
<p class="body_text"><br />
Snapshots of the team members were taken!<br />
</p><br />
<p class="body_text"><br />
The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.<br />
</p><br />
<p class="body_text"><br />
Alex contacted the Imperial iGEM team regarding an eventual collaboration. <br />
</p><br />
<br />
<p class="minor_title">6th August</p><br />
<p class="body_text"><br />
Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.<br />
</p><br />
<br />
<p class="minor_title">7th August</p><br />
<p class="body_text"><br />
Barbeque evening, venue Wilkins Roof Garden!<br />
</p><br />
<p class="body_text"><br />
Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)<br />
</p><br />
<br />
<p class="minor_title">8th August</p><br />
<p class="body_text"><br />
The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:<br />
</p><br />
<p class="body_text"><br />
i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly<br />
</p><br />
<p class="body_text"><br />
ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants. <br />
</p><br />
<br />
<p class="minor_title">9th August</p><br />
<p class="body_text"><br />
Project description is up on Wiki!<br />
</p><br />
<p class="body_text"><br />
Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm.<br />
The requested batch of biobricks arrived as glycerol stocks.<br />
</p><br />
<p class="body_text"><br />
The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested. <br />
</p><br />
<br />
<p class="minor_title">12th August</p><br />
<p class="body_text"><br />
We had a strategy chat at the lab with Darren. <br />
</p><br />
<p class="body_text"><br />
FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.<br />
The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.<br />
</p><br />
<p class="body_text"><br />
In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up. <br />
</p><br />
<p class="body_text"><br />
The website came to life today!<br />
</p><br />
<br />
<br />
<p class="minor_title">13th August</p><br />
<p class="body_text"><br />
Alex and Oran came up with the idea of a Creative writing competition. <br />
</p><br />
<p class="body_text"><br />
FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.<br />
</p><br />
<br />
<br />
<p class="minor_title">14th August</p><br />
<p class="body_text"><br />
The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.<br />
</p><br />
<p class="body_text"><br />
Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.<br />
</p><br />
<br />
<p class="minor_title">15th August</p><br />
<p class="body_text"><br />
Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design. <br />
</p><br />
<br />
<p class="minor_title">16th August</p><br />
<p class="body_text"><br />
Alex finished the essay on Neuroethics on which he has dedicated around 2 weeks of research.<br />
</p><br />
<br />
<p class="minor_title">19th August</p><br />
<p class="body_text"><br />
Alex advertised the writing competition on prizemagic.co.uk.<br />
</p><br />
<p class="body_text"><br />
Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.<br />
</p><br />
<br />
<p class="minor_title">20th August</p><br />
<p class="body_text"><br />
The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.<br />
</p><br />
<p class="body_text"><br />
The members’ Profiles are ready to be uploaded on wiki!<br />
</p><br />
<br />
<p class="minor_title">21th August</p><br />
<p class="body_text"><br />
The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.<br />
</p><br />
<p class="body_text"><br />
The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.<br />
</p><br />
<br />
<p class="minor_title">22th August</p><br />
<p class="body_text"><br />
The first Creative Competition Entry! Yey! Thank you!<br />
</p><br />
<p class="body_text"><br />
The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin. <br />
</p><br />
<p class="body_text"><br />
The Zeocin kill curve was derived, a concentration of 150 ug/ml was used.<br />
</p><br />
<br />
<p class="minor_title">23th August</p><br />
<p class="body_text"><br />
The main poster for the front page was finalised. Well done FYi!<br />
</p><br />
<p class="body_text"><br />
New submissions for the Creative writing! <br />
Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!<br />
</p><br />
<br />
<br />
<p class="minor_title">26th August</p><br />
<p class="body_text"><br />
The lab was closed today hence we all focused on the wiki content.<br />
</p><br />
<p class="body_text"><br />
The front page poster background - wasteland was completed.<br />
</p><br />
<br />
<br />
<p class="minor_title">27th August</p><br />
<p class="body_text"><br />
Weiling emailed Geneious and Eppendorf with regards to Sponsorship.<br />
</p><br />
<br />
<p class="minor_title">28th August</p><br />
<p class="body_text"><br />
The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.<br />
</p><br />
<p class="body_text"><br />
We met Darren at 4 pm in the lab to discuss about the biobrick processing.<br />
</p><br />
<br />
<p class="minor_title">29th August</p><br />
<p class="body_text"><br />
We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.<br />
</p><br />
<p class="body_text"><br />
Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.<br />
</p><br />
<br />
<p class="minor_title">30th August</p><br />
<p class="body_text"><br />
We uploaded the first samples of memories on the Memory Lane page.<br />
</p><br />
<br />
<br />
</div><br />
<br />
<p class="major_title">September</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st September</p><br />
<p class="body_text"><br />
The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.<br />
<br />
<p class="minor_title">2nd September</p><br />
<p class="body_text"><br />
We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.<br />
<br />
<p class="minor_title">3rd September</p><br />
<p class="body_text"><br />
We started to consider which type of poster would be the best for the Jamboree presentation.<br />
We met Darren at 4 pm to discuss about the cloning strategy for MMP9.<br />
</p><br />
<br />
<p class="minor_title">4th September</p><br />
<p class="body_text"><br />
We used SurveryMonkey in order to make a decision on who should present at the Jamboree. <br />
We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.<br />
</p><br />
<br />
<p class="minor_title">5th September</p><br />
<p class="body_text"><br />
We decided that the best option as the background colour for the T-shirts would be white.<br />
</p><br />
<br />
<p class="minor_title">6th September</p><br />
<p class="body_text"><br />
HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!<br />
<br />
Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.<br />
</p><br />
<br />
<p class="minor_title">9th September</p><br />
<p class="body_text"><br />
Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells. <br />
A new ligation for zeocin was prepared and competent cells were transformed with it.<br />
</p><br />
<br />
<p class="minor_title">10th September</p><br />
<p class="body_text"><br />
All the photos of the team members and supervisors were mounted on wiki.<br />
We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a positive control when minipreping.<br />
</p><br />
<br />
<p class="minor_title">11th September</p><br />
<p class="body_text"><br />
Intense work in the Bacterial Lab as the Biobrick Submission deadline is nigh.<br />
</p> <br />
<p class="body_text"><br />
Weiling sent further sponsorship proposals to GSK and New England Biolabs.<br />
</p><br />
<br />
<p class="minor_title">12th September</p><br />
<p class="body_text"><br />
We agreed on the final details for the T-shirts.<br />
</p><br />
<p class="body_text"><br />
Robin released the update on Modelling. Yey!<br />
</p><br />
<p class="body_text"><br />
Darren gave us some OneShot Top 10 competent cells from 2004 in order to continue with the transformations.<br />
</p><br />
<br />
<p class="minor_title">13th September</p><br />
<br />
<p class="minor_title">14th September</p><br />
<p class="body_text"><br />
We decided not to use K812014 biobrick anymore because of the inconsistent digestion. We're always obtaining 3 bands instead of 2 when digesting with EcoR1 and Pst1.<br />
<br />
<br />
<p class="minor_title">15th September</p><br />
<p class="body_text"><br />
After many minipreps of the stock of 4 transformations and subsequent digestions of these DNAs, we finally identified the ligated zeocin into pSB1C3 (origin, second ligation and transformation set).<br />
</p><br />
<br />
<p class="minor_title">16th September</p><br />
<p class="body_text"><br />
Weiling set ligations of MMP9 in pSB1C3 after pcr-ing it and digesting it with EcoR1, Pst1 and Dpn1.<br />
</p><br />
<br />
<p class="minor_title">17th September</p><br />
<br />
<p class="minor_title">18th September</p><br />
<p class="body_text"><br />
Work is being done on the presentation preparation. A first draft of the powerpoint was produced and people invited to give feedback on it.<br />
</p><br />
<br />
<p class="minor_title">19th September</p><br />
<p class="body_text"><br />
Bacteria Lab worked on maxipreping the recombinant zeocin plamid as well as on the MMP9 recombinant plasmid.<br />
</p><br />
<br />
<p class="minor_title">20th September</p><br />
<p class="body_text"><br />
Today is the deadline for sending our biobrick. Casey prepared for shipping and sent the zeocin biobrick.<br />
</p><br />
<br />
<p class="minor_title">23rd September</p><br />
<br />
<p class="minor_title">24thSeptember</p><br />
<p class="body_text"><br />
Transformation of HeLa cells with the recombinant zeocin plasmid was performed today under the assistance of Alex Kinna. Thanks Alex! <br />
</p><br />
<p class="body_text"><br />
This transformation was proven to be successful!<br />
</p><br />
<br />
<p class="minor_title">25th September</p><br />
<p class="body_text"><br />
The company to print our T-shirts was chosen. We're going with Image Scotland.<br />
</p><br />
<br />
<p class="minor_title">26th September</p><br />
<p class="body_text"><br />
Two representatives of Source Biosciences payed us a visit in the tissue culture lab at 2pm. They discussed transfection methods with us and advertised their reagents.<br />
</p><br />
<br />
<p class="minor_title">27th September</p><br />
<p class="body_text"><br />
Darren confirmed with us the funding for the trip to come! Friday, the 11th of October, in the afternoon, we're flying to Lyon!<br />
</p><br />
<br />
<p class="minor_title">30th September</p><br />
<br />
</div><br />
<br />
<p class="major_title">October</p><br />
<div class="full_row"> <br />
<div class="gap"><br />
</div><br />
<br />
<p class="minor_title">1st October</p><br />
<p class="body_text"><br />
Darren visited us in the Mammalian Lab and gave us the CMV-MMP9 control plasmid.<br />
</p><br />
<br />
<p class="minor_title">2nd October</p><br />
<p class="body_text"><br />
The entire team met Darren to rehearse the presentation for the Jamboree in Lyon.<br />
</p><br />
<br />
<p class="minor_title">3rd October</p><br />
<p class="body_text"><br />
Robin took charge of the collaboration on Modelling for Westminster iGEM team.<br />
</p><br />
<p class="body_text"><br />
FYi finalised the circuit drawing which was mounted on the Wiki.<br />
The digestion of cmv+MMP9 recombinant plasmid showed promising results.<br />
</p><br />
<br />
<p class="minor_title">4th October</p><br />
<p class="body_text"><br />
Today we received the T-Shirts. <br />
</p><br />
<p class="body_text"><br />
The team reunited at Robin's to make sure that everything is mounted and that the wiki is in order right before the Wiki Freeze at 4:59 am.<br />
</p><br />
<p class="body_text"> <br />
We also took advantage of this event and had our group photo taken all of us wearing our brand new Spotless mind T-shirts! We also included Stjohn's photo who wasn't able to be with us tonight but with whom we'll be finally reunited in Lyon! Yey!<br />
</p><br />
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</html></div>AlexBateshttp://2013.igem.org/Team:UCL/Modeling/BioinformaticsTeam:UCL/Modeling/Bioinformatics2013-10-05T01:50:48Z<p>AlexBates: </p>
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<p class="major_title">A BIOINFORMATICS APPROACH</p><br />
<p class="minor_title">Finding New Parts</p><br />
<p class="body_text"><br />
Bioinformatics creates and enhances methods for storing, retrieving, organising and analysing biological data. We decided to take a completely new approach in our dry lab work and look into bioinformatic approaches to studying <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a>. <br />
</p> <br />
<p class="body_text"><br />
The rationale behind this is simple. In order to make a genetic circuit in a synthetic biological construct as effective as possible in a medical application, we may need to target key dysfunctional genes within the problematic biological entity. There are many risk factors for AD and so predicting the key, ‘driver genes’, and the group of proteins with which they interact is invaluable in knowing what we want our construct to produce, in order to mitigate AD. The idea is that bioinformatics work can feed back into synthetic biology, and though we did not have the time to demonstrate this full circle, we feel bioinformatics can have a place in iGEM, helping teams to decide which dysfunctional genes to target in medical projects.</p><br />
<br />
<div class="small_image_left"><br />
<a href="https://static.igem.org/mediawiki/2013/0/03/Human_interactome.jpg" data-lightbox="image-1" title="The Human Interactome"><br />
<img src="https://static.igem.org/mediawiki/2013/0/03/Human_interactome.jpg"><br />
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<br />
<p class="body_text"><br />
<p class="minor_title">Bioinformatics and Alzheimer’s Disease</p> <br />
<p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. <br />
</p><p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>.<br />
</p><p class="body_text"><br />
In all pathologies, the most common way to predict driver genes is to target commonly recurrent genes. However, this approach misses misses rare altered genes which comprise the majority of genetic defects leading to, for example, carcinogenesis and arguably AD. This is partly because alterations in a single protein module can lead to the same disease phenotype. Thus, identification may best be attempted on a modular level. Yet it is also important to note correlation events between modules. Simply put, many rare gene alterations that influence the module they belong to and co-altered modules can collectively generate the disease pathology (Gu et al. 2013).<br />
<div class="gap"><br />
</div><br />
<p class="minor_title">Our Programme</p> <br />
<p class="body_text"><br />
Under the guidance and tutelage of <a href="http://bmm.cancerresearchuk.org/~cheng03/" target="_blank">Dr Tammy Cheng</a> from the <a href="http://bmm.cancerresearchuk.org/" target="_blank">Biomolecular Modelling (BMM) lab</a> at Cancer Research UK, team member <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Alexander Bates</a> coded in python a network analysis programme based on a method devised by Gu et al. and originally applied to the study of glioblastoma (brain cancer). The programme tries to reveal driver genes and co-altered functional modules for AD. The analysis procedure involves mapping altered genes (mutations, amplifications, repressions, etc.) in patient microRNA data to the protein interaction network (PIT), which currently accounts for 48,480 interactions between 10,982 human genes. This is termed the ‘AD altered network’, and is searched with the algorithm suggested by Gu et al. (which has been re-coded from scratch).<br />
</p><br />
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<p class="body_text"><br />
The programme builds up gene sets, two at a time, starting from two seed genes. These sets are termed 'modules'. Pairs of modules (‘G1’ and ‘G2’ in equation) are assumed to be co-altered if any gene within each module is altered in a proportion of AD sufferers, and genes between the modules are often altered together. For two modules, G1 and G2, we must calculate the probability, P, of observing than the number of the samples in the patient gene expression data that by chance simultaneously carry alterations in both gene sets. The gene expression data originates from post-mortem brain samples.<br />
</p><br />
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‘n’ is the total number of patient samples, ‘a’ is the number of patients with alterations in both G1 and G2, ‘b’ is the number of patients with alteration in just G1, ‘c’ is the number of patients with alterations in only G2, and ‘d’ is the number of patients with alterations in neither set. The co-altered score’ S, is defined below. A high score indicates that the two modules tend to be altered together in AD.<br />
</p><p class="body_text"><br />
Fig.1 depicts the searching algorithm. It searches and builds co-altered module pairs for the gene combinations within them that have the greatest co-alteration scores. In step 1, it methodically choose two seed genes from the AD altered network. The ellipsoids in the diagram denote direct interaction partners for these genes. These are added to the seeds to make temporary module pairs. The dashed line represents co-alteration. In step 2, the co-alteration score for each temporary module pair is calculated. Only the pair with the maximal S score is retained for subsequent searching. This maximal group becomes the new seeds group in step 3. In step 4, temporary modules are again derived, this time from step 3, and the maximum score is kept. In step 5, it must determine whether or not this group of genes is going to continue to expand. Each new addition save for the original two starting seeds is removed and S is recalculated. If in one of these configurations S becomes smaller, we loop through steps 3 to 5 again. Otherwise, if all combinations equate to the S value of the gene groups chosen from step 4, the process stops, having assumed that we have reached maximal module size for the two starting seeds.<br />
</p><br />
<p class="body_text"><br />
In other words, we try to build up gene sets within a module as large was we can, whilst with each new addition increasing the co-alteration score.<br />
</p><br />
<p class="body_text"><br />
We should end up with modules that frequently exhibit significant co-alteration in AD patients, and their gene products are therefore likely to be biochemically significant in the disease state.<br />
</p><p class="body_text"><br />
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<p class="minor_title">Results</p> <br />
<p class="body_text"><br />
Originally we planned, as previously suggested, to use the entirety of the human interactome to create an AD interactome and then run our programme in such a way as to build modules from this interactome. However, the estimated run time of the programme over-shot the iGEM 'wiki freeze' deadline. Therefore, we used the expression data for 311 hub genes, whose proteins are points of high connectivity in the human interactome, across 62 modules defined by Zhang et al., and searched for the hub genes combinations that produced the greatest co-alteration scores. The 62 modules are named after colours. <br />
</p><br />
<p class="body_text"><br />
<b>Module groups: </b> <a href="https://static.igem.org/mediawiki/2013/e/ec/AlzModules.txt" target="_blank">AlzModules.py</a><br />
<p class="body_text"><br />
<b>Hub expression data:</b> <a href="https://static.igem.org/mediawiki/2013/7/7a/ALzData2.txt" target="_blank">AlzData.py</a><br />
</p><br />
<p class="body_text"><br />
<b>Module matrix:</b> <a href="https://static.igem.org/mediawiki/2013/5/5f/AlzList.txt" target="_blank">AlzMatrix.py</a><br />
</p><br />
<p class="body_text"><br />
The code for our network analysis programme can be found <a href="https://static.igem.org/mediawiki/2013/4/40/Alex4.txt" target="_blank">here</a>. It needs to be converted to a .py file to be used. Please note that the output is given as a set of numbers that as assigned to genes. For example, the final output for the data we ran can be found <a href="https://static.igem.org/mediawiki/2013/0/0f/AlzFinal.txt" target="_blank">here</a>.<br />
</p><br />
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<table><br />
<th><p class="citation_text">Fig.1 Histogram showing the frequency of gene sets by co-alteration score.</p></th><br />
</table><br />
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<p class="body_text"><br />
We used the output of our programme to produce a histogram, which shows that the frequency of gene combinations falls exponentially with increasing co-alteration score This suggests that a significant few combinations are regularly co-altered in Alzheimer's disease, in modules that may help drive the disease state. Because we are only looking at which hub genes within modules, we are most interested in what modules are co-altered in the high score end of the histogram, and not the hub genes specifically.</p><br />
<p class="body_text"><br />
Below, Fig.2 shows the twenty gene set pairs between two modules, which yielded the greatest co-alteration score. The module pair with the highest score, and that recurs most frequently in the top twenty, are the 'Khaki' and 'Honey Dew' modules. The most enriched functional category of the khaki module is the biosynthesis of a neurotransmitter called GABA. GABA is responsible for neuronal excitability and muscle tone. The Honey Dew module is primarily involved in muscle contraction, though the hub genes AHCYL1 and C9orf61 are thought to be involved in inositol signaling and are possibly associated with another brain condition, bi-polar disorder. However, since the gene expression data is from generally older patients, given the profile of AD, these muscle associated modules may be altered together because of changing muscle usage with age (there is no muscle in the brain but this may represent brain cell structural integrity). Both of these modules have almost 100% of their total brain gene expression in the prefrontal cortex, and area known to be heavily impacted in AD, causing cognitive and intellectual damage. This suggests that our genetic circuit could be adapted to target signaling mechanisms in this area.</p><br />
<br />
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<br />
<table><br />
<th><p class="citation_text">Fig.2 Table of the top 20 gene combinations and their modules by co-alteration score.</p></th><br />
</table><br />
<table><br />
<tr><br />
<th>Module Name and Gene Set</th><br />
<th>Module Name and Gene Set</th><br />
<th>Co-alteration Score</th><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>20.39 </td><br />
<tr><br />
<td>SLC15A2, FXYD1</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.73 </td><br />
<tr><br />
<td>GJA1, FXYD1</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.37 </td><br />
<tr><br />
<td>GJA1, FXYD1, ATP13A4</td><br />
<td>C20orf141, RFX4, AHCYL1, DGCR6</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.99 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.81 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>17.69 </td><br />
<tr><br />
<td>GJA1, FXYD1, SLC15A2</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>17.57 </td><br />
<tr><br />
<td>RRM2, NM_022346, FAM64A</td><br />
<td>OR4F5, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>17.49 </td><br />
<tr><br />
<td>DYNC2LI1, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP, CRYBA2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>16.78 </td><br />
<tr><br />
<td>CIRBP, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.64 </td><br />
<tr><br />
<td>RRM2, NMMR, FAM64A</td><br />
<td>KRTHB4, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.47 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.46 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr> <br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Forestgreen</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.43 </td><br />
<tr><br />
<td>IFITM3, CSDA</td><br />
<td>CSDA</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.38 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.27 </td><br />
<tr><br />
<td>FXYD1, ATP13A4, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.25 </td><br />
<tr><br />
<td>FXYD1, ATP13A4</td><br />
<td>DGCR6, AHCYL1, C20orf141, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Gold 2</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.21 </td><br />
<tr><br />
<td>TUBB2B, NM_178525</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.04 </td><br />
<tr><br />
<td>SPON1, FXYD1, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
</table> <br />
<br />
</p><br />
<p class="body_text"><br />
The second highest scoring module pair, and the second most frequent in the top twenty, are 'Turquoise' and 'Cyan'. The former is primarily involved with NAD(P) homeostasis, and so is significant in cells' metabolism, while the genes in the later mainly play a role in vasculature development. This suggests that co-alteration in genes involved within these two modules could impact cell vitality and trophic support and help cause AD. This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
The third highest scoring module pair, and the third most frequent in the top twenty, are 'Green 4' and 'Yellow 3'. Green 4 is involved in cell cycle regulation, and area that has already been targeted by our circuit, which produces BDNF to help avoid chromosomal division in the neurons of AD patients. Yellow 3 is associated with the peripheral nervous system. Co-alteration here may again be indicative of gene expression changes with age, and its link with Green 4 may suggest that this is to do with a deficiency in cell division, regeneration and growth, but this is not directly related to AD, although hub genes like GRAP do play a role in cytoplasmic signaling in cells including neurons and glia, This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
Other module pairs that feature in the top twenty include 'Wheat' and 'Turqouise', 'Forestgreen' and 'Cyan' and 'Gold 2' and 'Honey Dew'. Wheat is involved in protein folding and responses to unfolded and mis-folded protein. This is significant because incorrectly formed and folded amyloid is strongly associated with the progression of AD. This is something out circuit already seeks to address, but by targeting elements of the 'Wheat' module and similar modules it could aim to avoid mis-creation in the first place, and the nucleation of other mis-folded proteins. Forestgreen is involved in immune functions, which implicates microglia and the cellular response to inflammation in neurons - factors our circuit already tries to help address by acting to prevent neuroinflammation. Its association with Cyan could imply that negative inflammatory effects may be inked with brain vasculature in AD. Gold 2 is associated with the cytoskeleton and axonal cytoskeletal control.In AD, the formation of plaques and protein tangles disrupts the cytoskeleton and perturb axonal connections, engendering cell death. Our circuit tries to target this already by removing the plaques, but perhaps a future improvement should to be to create an element capable to supporting a healthy cytoskeleton or able to remove cytoskeletal protein tangles. Its association with Honey Dew, however, could point to unusual gene expression in this module being due to the lessened use of muscle in old age.</p><br />
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</html></div>AlexBateshttp://2013.igem.org/File:Alex4.txtFile:Alex4.txt2013-10-05T01:49:25Z<p>AlexBates: </p>
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<div></div>AlexBateshttp://2013.igem.org/Team:UCL/Modeling/BioinformaticsTeam:UCL/Modeling/Bioinformatics2013-10-05T01:45:58Z<p>AlexBates: </p>
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<p class="major_title">A BIOINFORMATICS APPROACH</p><br />
<p class="minor_title">Finding New Parts</p><br />
<p class="body_text"><br />
Bioinformatics creates and enhances methods for storing, retrieving, organising and analysing biological data. We decided to take a completely new approach in our dry lab work and look into bioinformatic approaches to studying <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s disease (AD)</a>. <br />
</p> <br />
<p class="body_text"><br />
The rationale behind this is simple. In order to make a genetic circuit in a synthetic biological construct as effective as possible in a medical application, we may need to target key dysfunctional genes within the problematic biological entity. There are many risk factors for AD and so predicting the key, ‘driver genes’, and the group of proteins with which they interact is invaluable in knowing what we want our construct to produce, in order to mitigate AD. The idea is that bioinformatics work can feed back into synthetic biology, and though we did not have the time to demonstrate this full circle, we feel bioinformatics can have a place in iGEM, helping teams to decide which dysfunctional genes to target in medical projects.</p><br />
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<a href="https://static.igem.org/mediawiki/2013/0/03/Human_interactome.jpg" data-lightbox="image-1" title="The Human Interactome"><br />
<img src="https://static.igem.org/mediawiki/2013/0/03/Human_interactome.jpg"><br />
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<p class="body_text"><br />
<p class="minor_title">Bioinformatics and Alzheimer’s Disease</p> <br />
<p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. <br />
</p><p class="body_text"><br />
Recent progress in characterising AD has lead to the identification of dozens of highly interconnected genetic risk factors, yet it is likely that many more remain undiscovered <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044851/" target="_blank">(Soler-Lopez et al. 2011)</a> and the elucidation of their roles in AD could prove pivotal in beating the condition. AD is genetically complex, linked with many defects both mutational or of susceptibility. These defects produce alterations in the molecular interactions of cellular pathways, the collective effect of which may be gauged through the structure of the protein network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>. In other words, there is a strong link between protein connectivity and the disease phenotype. AD arises from the downstream interplay between genetic and non-genetic alterations in the human protein interaction network <a href="http://www.sciencedirect.com/science/article/pii/S0092867413003875" target="_blank">(Zhang et al. 2013)</a>.<br />
</p><p class="body_text"><br />
In all pathologies, the most common way to predict driver genes is to target commonly recurrent genes. However, this approach misses misses rare altered genes which comprise the majority of genetic defects leading to, for example, carcinogenesis and arguably AD. This is partly because alterations in a single protein module can lead to the same disease phenotype. Thus, identification may best be attempted on a modular level. Yet it is also important to note correlation events between modules. Simply put, many rare gene alterations that influence the module they belong to and co-altered modules can collectively generate the disease pathology (Gu et al. 2013).<br />
<div class="gap"><br />
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<p class="minor_title">Our Programme</p> <br />
<p class="body_text"><br />
Under the guidance and tutelage of <a href="http://bmm.cancerresearchuk.org/~cheng03/" target="_blank">Dr Tammy Cheng</a> from the <a href="http://bmm.cancerresearchuk.org/" target="_blank">Biomolecular Modelling (BMM) lab</a> at Cancer Research UK, team member <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Alexander Bates</a> coded in python a network analysis programme based on a method devised by Gu et al. and originally applied to the study of glioblastoma (brain cancer). The programme tries to reveal driver genes and co-altered functional modules for AD. The analysis procedure involves mapping altered genes (mutations, amplifications, repressions, etc.) in patient microRNA data to the protein interaction network (PIT), which currently accounts for 48,480 interactions between 10,982 human genes. This is termed the ‘AD altered network’, and is searched with the algorithm suggested by Gu et al. (which has been re-coded from scratch).<br />
</p><br />
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The programme builds up gene sets, two at a time, starting from two seed genes. These sets are termed 'modules'. Pairs of modules (‘G1’ and ‘G2’ in equation) are assumed to be co-altered if any gene within each module is altered in a proportion of AD sufferers, and genes between the modules are often altered together. For two modules, G1 and G2, we must calculate the probability, P, of observing than the number of the samples in the patient gene expression data that by chance simultaneously carry alterations in both gene sets. The gene expression data originates from post-mortem brain samples.<br />
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‘n’ is the total number of patient samples, ‘a’ is the number of patients with alterations in both G1 and G2, ‘b’ is the number of patients with alteration in just G1, ‘c’ is the number of patients with alterations in only G2, and ‘d’ is the number of patients with alterations in neither set. The co-altered score’ S, is defined below. A high score indicates that the two modules tend to be altered together in AD.<br />
</p><p class="body_text"><br />
Fig.1 depicts the searching algorithm. It searches and builds co-altered module pairs for the gene combinations within them that have the greatest co-alteration scores. In step 1, it methodically choose two seed genes from the AD altered network. The ellipsoids in the diagram denote direct interaction partners for these genes. These are added to the seeds to make temporary module pairs. The dashed line represents co-alteration. In step 2, the co-alteration score for each temporary module pair is calculated. Only the pair with the maximal S score is retained for subsequent searching. This maximal group becomes the new seeds group in step 3. In step 4, temporary modules are again derived, this time from step 3, and the maximum score is kept. In step 5, it must determine whether or not this group of genes is going to continue to expand. Each new addition save for the original two starting seeds is removed and S is recalculated. If in one of these configurations S becomes smaller, we loop through steps 3 to 5 again. Otherwise, if all combinations equate to the S value of the gene groups chosen from step 4, the process stops, having assumed that we have reached maximal module size for the two starting seeds.<br />
</p><br />
<p class="body_text"><br />
In other words, we try to build up gene sets within a module as large was we can, whilst with each new addition increasing the co-alteration score.<br />
</p><br />
<p class="body_text"><br />
The P-values of the co-altered modules this algorithm identifies are modified by the Benjamini–Hochberg procedure and those with an FDR < 10% are kept. If a pair of co-altered modules share more than 50% of their genes with another pair, the one with the lowest S score is discarded. We should be left with modules that frequently exhibit significant co-alteration in AD patients, and their gene products are therefore likely to be biochemically significant in the disease state.<br />
</p><p class="body_text"><br />
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<p class="minor_title">Results</p> <br />
<p class="body_text"><br />
Originally we planned, as previously suggested, to use the entirety of the human interactome to create an AD interactome and then run our programme in such a way as to build modules from this interactome. However, the estimated run time of the programme over-shot the iGEM 'wiki freeze' deadline. Therefore, we used the expression data for 311 hub genes, whose proteins are points of high connectivity in the human interactome, across 62 modules defined by Zhang et al., and searched for the hub genes combinations that produced the greatest co-alteration scores. The 62 modules are named after colours. <br />
</p><br />
<p class="body_text"><br />
<b>Module groups: </b> <a href="https://static.igem.org/mediawiki/2013/e/ec/AlzModules.txt" target="_blank">AlzModules.py</a><br />
<p class="body_text"><br />
<b>Hub expression data:</b> <a href="https://static.igem.org/mediawiki/2013/7/7a/ALzData2.txt" target="_blank">AlzData.py</a><br />
</p><br />
<p class="body_text"><br />
<b>Module matrix:</b> <a href="https://static.igem.org/mediawiki/2013/5/5f/AlzList.txt" target="_blank">AlzMatrix.py</a><br />
</p><br />
<p class="body_text"><br />
The code for our network analysis programme can be found <a href="https://static.igem.org/mediawiki/2013/7/7a/ALzData2.txt" target="_blank">here</a>. Please note that the output is given as a set of numbers that as assigned to genes. For example, the final output for the data we ran can be found <a href="https://static.igem.org/mediawiki/2013/0/0f/AlzFinal.txt" target="_blank">here</a>.<br />
</p><br />
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<table><br />
<th><p class="citation_text">Fig.1 Histogram showing the frequency of gene sets by co-alteration score.</p></th><br />
</table><br />
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<p class="body_text"><br />
We used the output of our programme to produce a histogram, which shows that the frequency of gene combinations falls exponentially with increasing co-alteration score This suggests that a significant few combinations are regularly co-altered in Alzheimer's disease, in modules that may help drive the disease state. Because we are only looking at which hub genes within modules, we are most interested in what modules are co-altered in the high score end of the histogram, and not the hub genes specifically.</p><br />
<p class="body_text"><br />
Below, Fig.2 shows the twenty gene set pairs between two modules, which yielded the greatest co-alteration score. The module pair with the highest score, and that recurs most frequently in the top twenty, are the 'Khaki' and 'Honey Dew' modules. The most enriched functional category of the khaki module is the biosynthesis of a neurotransmitter called GABA. GABA is responsible for neuronal excitability and muscle tone. The Honey Dew module is primarily involved in muscle contraction, though the hub genes AHCYL1 and C9orf61 are thought to be involved in inositol signaling and are possibly associated with another brain condition, bi-polar disorder. However, since the gene expression data is from generally older patients, given the profile of AD, these muscle associated modules may be altered together because of changing muscle usage with age (there is no muscle in the brain but this may represent brain cell structural integrity). Both of these modules have almost 100% of their total brain gene expression in the prefrontal cortex, and area known to be heavily impacted in AD, causing cognitive and intellectual damage. This suggests that our genetic circuit could be adapted to target signaling mechanisms in this area.</p><br />
<br />
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<table><br />
<th><p class="citation_text">Fig.2 Table of the top 20 gene combinations and their modules by co-alteration score.</p></th><br />
</table><br />
<table><br />
<tr><br />
<th>Module Name and Gene Set</th><br />
<th>Module Name and Gene Set</th><br />
<th>Co-alteration Score</th><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>20.39 </td><br />
<tr><br />
<td>SLC15A2, FXYD1</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.73 </td><br />
<tr><br />
<td>GJA1, FXYD1</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>19.37 </td><br />
<tr><br />
<td>GJA1, FXYD1, ATP13A4</td><br />
<td>C20orf141, RFX4, AHCYL1, DGCR6</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.99 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>18.81 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2, CDK2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>17.69 </td><br />
<tr><br />
<td>GJA1, FXYD1, SLC15A2</td><br />
<td>RFX4, AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>17.57 </td><br />
<tr><br />
<td>RRM2, NM_022346, FAM64A</td><br />
<td>OR4F5, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>17.49 </td><br />
<tr><br />
<td>DYNC2LI1, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.95 </td><br />
<tr><br />
<td>HMMR</td><br />
<td>OR4F5, GRAP, CRYBA2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Wheat</b></td><br />
<td>16.78 </td><br />
<tr><br />
<td>CIRBP, RBM4</td><br />
<td>AF087999</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Green 4</b></td><br />
<td><b>Yellow 3</b></td><br />
<td>16.64 </td><br />
<tr><br />
<td>RRM2, NMMR, FAM64A</td><br />
<td>KRTHB4, GRAP, XM_166973</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.47 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.46 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>Contig47252_RC, IFITM2, CDK2</td><br />
</tr> <br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Forestgreen</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.43 </td><br />
<tr><br />
<td>IFITM3, CSDA</td><br />
<td>CSDA</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Turquoise</b></td><br />
<td><b>Cyan</b></td><br />
<td>16.38 </td><br />
<tr><br />
<td>DYNC2LI1, CIRBP, ACRC, RCC1, RBM4</td><br />
<td>ENST00000289005, Contig47252_RC, IFITM2</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.27 </td><br />
<tr><br />
<td>FXYD1, ATP13A4, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.25 </td><br />
<tr><br />
<td>FXYD1, ATP13A4</td><br />
<td>DGCR6, AHCYL1, C20orf141, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Gold 2</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.21 </td><br />
<tr><br />
<td>TUBB2B, NM_178525</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
<tr><br />
<td></td><br />
</tr><br />
<td><b>Khaki</b></td><br />
<td><b>Honey Dew</b></td><br />
<td>16.04 </td><br />
<tr><br />
<td>SPON1, FXYD1, SLC15A2</td><br />
<td>AHCYL1, C9orf61</td><br />
</tr><br />
</table> <br />
<br />
</p><br />
<p class="body_text"><br />
The second highest scoring module pair, and the second most frequent in the top twenty, are 'Turquoise' and 'Cyan'. The former is primarily involved with NAD(P) homeostasis, and so is significant in cells' metabolism, while the genes in the later mainly play a role in vasculature development. This suggests that co-alteration in genes involved within these two modules could impact cell vitality and trophic support and help cause AD. This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
The third highest scoring module pair, and the third most frequent in the top twenty, are 'Green 4' and 'Yellow 3'. Green 4 is involved in cell cycle regulation, and area that has already been targeted by our circuit, which produces BDNF to help avoid chromosomal division in the neurons of AD patients. Yellow 3 is associated with the peripheral nervous system. Co-alteration here may again be indicative of gene expression changes with age, and its link with Green 4 may suggest that this is to do with a deficiency in cell division, regeneration and growth, but this is not directly related to AD, although hub genes like GRAP do play a role in cytoplasmic signaling in cells including neurons and glia, This suggests that our circuit could be improved by being adapted to help maintain general cell health and energy supply in the brain. </p><br />
<p class="body_text"><br />
Other module pairs that feature in the top twenty include 'Wheat' and 'Turqouise', 'Forestgreen' and 'Cyan' and 'Gold 2' and 'Honey Dew'. Wheat is involved in protein folding and responses to unfolded and mis-folded protein. This is significant because incorrectly formed and folded amyloid is strongly associated with the progression of AD. This is something out circuit already seeks to address, but by targeting elements of the 'Wheat' module and similar modules it could aim to avoid mis-creation in the first place, and the nucleation of other mis-folded proteins. Forestgreen is involved in immune functions, which implicates microglia and the cellular response to inflammation in neurons - factors our circuit already tries to help address by acting to prevent neuroinflammation. Its association with Cyan could imply that negative inflammatory effects may be inked with brain vasculature in AD. Gold 2 is associated with the cytoskeleton and axonal cytoskeletal control.In AD, the formation of plaques and protein tangles disrupts the cytoskeleton and perturb axonal connections, engendering cell death. Our circuit tries to target this already by removing the plaques, but perhaps a future improvement should to be to create an element capable to supporting a healthy cytoskeleton or able to remove cytoskeletal protein tangles. Its association with Honey Dew, however, could point to unusual gene expression in this module being due to the lessened use of muscle in old age.</p><br />
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