http://2013.igem.org/wiki/index.php?title=Special:Contributions/Andykecheng&feed=atom&limit=50&target=Andykecheng&year=&month=2013.igem.org - User contributions [en]2024-03-28T12:20:06ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UCL/Team/ProfileTeam:UCL/Team/Profile2013-10-05T03:58:29Z<p>Andykecheng: </p>
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b> MEng Biochemical Engineering. </b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Practice/NeuroethicsTeam:UCL/Practice/Neuroethics2013-10-05T03:55:55Z<p>Andykecheng: </p>
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<p class="major_title">THE NEUROETHICS REPORT</p><br />
<p class="minor_title">Why Look At Neuroethics?</p><br />
<p class="body_text"><br />
Our <a href="https://2013.igem.org/Team:UCL/Project" target="_blank"> project</a> deals with an idea which may seem, on the face of it, frightening to some; the insertion of modified brain cells, <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank"> microglia</a>, to try and alleviate <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank"> Alzheimer's disease (AD)</a>. Although more similar to a macrophage than a neuron, engineering microglial cells represents both a scientific and an ethical challenge, not least because it seems like the stuff of <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank"> zombie B-movies</a>. After all, using microglia to halt the progression of AD, and therefore cognitive loss, by dissolving senile plaques is only one philosophical step (albeit very many scientific steps) from a genetic system for cognitive gain, so the implications of our project stretch past medical bioethics. In the interests of assessing the feasibility of the project in <a href="http://www.sciencedirect.com/science/article/pii/S1364661304002955" target="_blank"> social terms</a>, we are producing this report dealing with the attitudes and <a href=http://www.nature.com/neuro/journal/v5/n11/full/nn1102-1123.html" target="_blank">neuroethics</a> of the potential use of neuro-genetic engineering in medicine, therapy and enhancement technology, as well as expounding a little on some of the scientific concepts behind various approaches.<br />
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<p class="minor_title">The Essay</p><br />
<p class="body_text"><br />
In a comprehensive report, team member <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Alexander Bates</a> takes a look at the medical ethics, the neuroethics and both the plausible and <a href="http://www.sciencedirect.com/science/article/pii/S0306987708002673" target="_blank"> fanciful</a> neuroscientific applications of synthetic biology: <p class="body_text"><b><a href="https://static.igem.org/mediawiki/2013/7/7c/Neuroethics_Report.pdf" target="_blank">Neuro-Genethics Report.PDF</a></p><br />
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<p class="minor_title">Read On Our Site</p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay1" target="_blank">Introduction: Medicine and Synthetic Biology</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay2" target="_blank">Medical Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay3" target="_blank">Therapeutic Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay4" target="_blank">Enhancement Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay5" target="_blank">The Core of the Neuroethical Debate</a></p><br />
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<p class="body_text"><b><a href="https://2013.igem.org/Team:UCL/Practice/Essay6" target="_blank">Conclusion</a></p><br />
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<p class="body_text"><b>Alex Bates</b> </p><br />
<p class="body_text">Our project is, as yet, highly theoretical, but it's implications lead us to one of the most fundamental questions in life: what is it to be human? Only once in our history has the human existence been radically redefined - at the origin on mankind, the transition from animals to intelligent, self-conscious beings. We are, perhaps, moving towards the frontier of another transition - the ability to induce dramatic changes in our consciousness at will. The question, "Should we genetically engineer the brain?" essentially asks, do we want to, or even have the right to, fundamentally redefine our existence for only the second time in our history. </p><br />
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<p class="body_text"><b>Ruxi Comisel </b></p><br />
<p class="body_text">I agree with the use of genetic engineering as part of a therapy provided that the only point of using it on the brain or in other parts of the human body is to alleviate the disastrous effect of disease on human integrity.<br />
I believe that the public should not reject this therapy as long as it is an available alternative and it can be used safely and under strict legal regulation, so that only the patients in advanced/terminal stages of suffering can benefit from it. <br />
On the other hand, I strongly oppose using genetic engineering in the context of patients who can benefit from other means of therapy known to be successful for the stages of disease they are at.</p><br />
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<p class="body_text"><b>Tom Johnson</b></p><br />
<p class="body_text">Genetic Engineering has been around for a while, but it has typically been associated with crops rather than people. If GM crops are questioned by the public then surely we need to look long and hard at how we will influence sentient beings. Unfair advantages could be had for the rich - people could effectively buy intelligence etc. which could divide the rich - poor barrier even further. <br />
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<p class="body_text"><b>Andy Cheng</b></p><br />
<p class="body_text">I personally believe genetic engineering is an amazing tool to program biological systems to perform tasks. However, the introduction of genetically engineered cells appear somewhat disturbing. We have to be able to prove these foreign cells would not interfere with the integrity of the mind. <br />
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<p class="body_text"><b>Oran Maguire</b></p><br />
<p class="body_text">My feelings about Synthetic Biology as a whole are quite confused. There are a huge number of potential applicaions which are capable of impacting on every part of our lives. These could come off very well or very badly for us. I think that the objections which are grounded in the importance of unaltered life and identity do not convince me. What does make me cautious about this technlogy is the potential for environmental hazards, and its potential to be socioeconomically divisive. Who knows how that will pan out. Right now, I get the impression that the way these projects are frequently presented, largely by young and the technically gifted students, will seem rather hubristic to many people looking in from the outside. Anyone aged 50 or under has every reason to take these extraordinary developments rather gravely, so to call projects such as these "cool" will ultimately strike a bad chord, and it will set people's opinions about Synthetic Biology prematurely.<br />
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<br />
<p class="major_title">THE NEUROETHICS REPORT</p><br />
<p class="minor_title">Why Look At Neuroethics?</p><br />
<p class="body_text"><br />
Our <a href="https://2013.igem.org/Team:UCL/Project" target="_blank"> project</a> deals with an idea which may seem, on the face of it, frightening to some; the insertion of modified brain cells, <a href="https://2013.igem.org/Team:UCL/Background/Microglia" target="_blank"> microglia</a>, to try and alleviate <a href="https://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank"> Alzheimer's disease (AD)</a>. Although more similar to a macrophage than a neuron, engineering microglial cells represents both a scientific and an ethical challenge, not least because it seems like the stuff of <a href="https://2013.igem.org/Team:UCL/Practice/Creative" target="_blank"> zombie B-movies</a>. After all, using microglia to halt the progression of AD, and therefore cognitive loss, by dissolving senile plaques is only one philosophical step (albeit very many scientific steps) from a genetic system for cognitive gain, so the implications of our project stretch past medical bioethics. In the interests of assessing the feasibility of the project in <a href="http://www.sciencedirect.com/science/article/pii/S1364661304002955" target="_blank"> social terms</a>, we are producing this report dealing with the attitudes and <a href=http://www.nature.com/neuro/journal/v5/n11/full/nn1102-1123.html" target="_blank">neuroethics</a> of the potential use of neuro-genetic engineering in medicine, therapy and enhancement technology, as well as expounding a little on some of the scientific concepts behind various approaches.<br />
</p><br />
<br />
<br />
<p class="minor_title">The Essay</p><br />
<p class="body_text"><br />
In a comprehensive report, team member <a href="https://2013.igem.org/Team:UCL/Team/Profile" target="_blank">Alexander Bates</a> takes a look at the medical ethics, the neuroethics and both the plausible and <a href="http://www.sciencedirect.com/science/article/pii/S0306987708002673" target="_blank"> fanciful</a> neuroscientific applications of synthetic biology: <p class="body_text"><b><a href="https://static.igem.org/mediawiki/2013/7/7c/Neuroethics_Report.pdf" target="_blank">Neuro-Genethics Report.PDF</a></p><br />
</p><br />
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<p class="minor_title">Read On Our Site</p><br />
<br />
<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay1" target="_blank">Introduction: Medicine and Synthetic Biology</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay2" target="_blank">Medical Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay3" target="_blank">Therapeutic Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay4" target="_blank">Enhancement Neuro-Genetic Engineering</a></p><br />
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<p class="body_text"><b> <a href="https://2013.igem.org/Team:UCL/Practice/Essay5" target="_blank">The Core of the Neuroethical Debate</a></p><br />
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<p class="body_text"><b><a href="https://2013.igem.org/Team:UCL/Practice/Essay6" target="_blank">Conclusion</a></p><br />
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<p class="body_text"><b><a href="https://2013.igem.org/Team:UCL/Practice/Essay7" target="_blank">Bibliography</a></p><br />
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<p class="minor_title">Team member's opinions on Neuroethics</p><br />
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<p class="body_text"><b>Alex Bates</b> </p><br />
<p class="body_text">Our project is, as yet, highly theoretical, but it's implications lead us to one of the most fundamental questions in life: what is it to be human? Only once in our history has the human existence been radically redefined - at the origin on mankind, the transition from animals to intelligent, self-conscious beings. We are, perhaps, moving towards the frontier of another transition - the ability to induce dramatic changes in our consciousness at will. The question, "Should we genetically engineer the brain?" essentially asks, do we want to, or even have the right to, fundamentally redefine our existence for only the second time in our history. </p><br />
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<p class="body_text"><b>Ruxi Comisel </b></p><br />
<p class="body_text">I agree with the use of genetic engineering as part of a therapy provided that the only point of using it on the brain or in other parts of the human body is to alleviate the disastrous effect of disease on human integrity.<br />
I believe that the public should not reject this therapy as long as it is an available alternative and it can be used safely and under strict legal regulation, so that only the patients in advanced/terminal stages of suffering can benefit from it. <br />
On the other hand, I strongly oppose using genetic engineering in the context of patients who can benefit from other means of therapy known to be successful for the stages of disease they are at.</p><br />
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<p class="body_text"><b>Tom Johnson</b></p><br />
<p class="body_text">Genetic Engineering has been around for a while, but it has typically been associated with crops rather than people. If GM crops are questioned by the public then surely we need to look long and hard at how we will influence sentient beings. Unfair advantages could be had for the rich - people could effectively buy intelligence etc. which could divide the rich - poor barrier even further. <br />
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<p class="body_text"><b>Andy Cheng</b></p><br />
<p class="body_text">I personally believe genetic engineering is an amazing tool to program biological systems to perform tasks. However, the introduction of genetically engineered cells appear somewhat disturbing. We have to be able to prove these foreign cells would not interfere with integrity of the mind. <br />
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<p class="body_text"><b>Oran Maguire</b></p><br />
<p class="body_text">My feelings about Synthetic Biology as a whole are quite confused. There are a huge number of potential applicaions which are capable of impacting on every part of our lives. These could come off very well or very badly for us. I think that the objections which are grounded in the importance of unaltered life and identity do not convince me. What does make me cautious about this technlogy is the potential for environmental hazards, and its potential to be socioeconomically divisive. Who knows how that will pan out. Right now, I get the impression that the way these projects are frequently presented, largely by young and the technically gifted students, will seem rather hubristic to many people looking in from the outside. Anyone aged 50 or under has every reason to take these extraordinary developments rather gravely, so to call projects such as these "cool" will ultimately strike a bad chord, and it will set people's opinions about Synthetic Biology prematurely.<br />
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b> MEng Biochemical Engineering. </b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering. </b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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<p class="major_title">SUPERVISORS</p><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 />
<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 />
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</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a0/Philipp_profile.jpg');"><br />
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<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 />
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<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>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 />
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</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>Dr. Howard Boland</b> is a multidisciplinary practitioner with a background in science and art. Howard is the artistic director and found of the organisation C-Lab. He holds an interest in contemporary arts and is very active in "Art from Synthetic Biology". He advised us on how to improve our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event.</p><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Shirley Nurock</b> is the Coordinator of the London Area Research Network in Alzheimer's Society. She holds experience as a carer and researcher in Alzheimer's Disease. She was a main speaker at our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event, where she underlined the desperation for a new way to tackle this tragic disease and warned about the ethics of over hyping our treatment.<br />
</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<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 />
</p><br />
<p class="body_text"><br />
<b>Annie Wei</b> is a PhD student of the UCL Biochemical Engineering department who offered us supervision and support with lab work<br />
</div><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 />
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</body><br />
</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T03:27:41Z<p>Andykecheng: </p>
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<p class="major_title">SUPERVISORS</p><br />
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<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/1/10/Darren_profile.jpg');"><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 />
<br />
</div><br />
<br />
<div class="col_1"><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 />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/a/a0/Philipp_profile.jpg');"><br />
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<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>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>Dr. Howard Boland</b> is a multidisciplinary practitioner with a background in science and art. Howard is the artistic director and found of the organisation C-Lab. He holds an interest in contemporary arts and is very active in "Art from Synthetic Biology". He advised us on how to improve our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event.</p><br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="body_text"><b>Shirley Nurock</b> is the Coordinator of the London Area Research Network in Alzheimer's Society. She holds experience as a carer and researcher in Alzheimer's Disease. She was a main speaker at our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event, where she underlined the desperation for a new way to tackle this tragic disease and warned about the ethics of over hyping our treatment.<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 />
</p><br />
<p class="body_text"><br />
<b>Annie Wei</b> is a PhD student of the UCL Biochemical Engineering department who offered us supervision and support with lab work<br />
</div><br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<!-- END CONTENT ------------------------------------------------------------------------------------------------------><br />
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</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 />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/AttributionsTeam:UCL/Team/Attributions2013-10-05T03:26:19Z<p>Andykecheng: </p>
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<p class="major_title">SUPERVISORS</p><br />
<div class="gap"><br />
</div><br />
<div class="row_small"><br />
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<div class="col_1" style="background-image:url('https://static.igem.org/mediawiki/2013/1/10/Darren_profile.jpg');"><br />
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<div class="col_1"><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 />
<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 />
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<div class="gap"><br />
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<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 />
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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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<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 />
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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 />
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<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 />
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<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 />
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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>Dr. Howard Boland</b> is a multidisciplinary practitioner with a background in science and art. Howard is the artistic director and found of the organisation C-Lab. He holds an interest in contemporary arts and is very active in "Art from Synthetic Biology". He advised us on hold our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event.</p><br />
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<p class="body_text"><b>Shirley Nurock</b> is the Coordinator of the London Area Research Network in Alzheimer's Society. She holds experience as a carer and researcher in Alzheimer's Disease. She was a main speaker at our <a href="https://2013.igem.org/Team:UCL/Debate" target="_blank">Speed debate</a> event, where she underlined the desperation for a new way to tackle this tragic disease and warned about the ethics of over hyping our treatment.<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 />
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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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<b>Annie Wei</b> is a PhD student of the UCL Biochemical Engineering department who offered us supervision and support with lab work<br />
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b>A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/ProfileTeam:UCL/Team/Profile2013-10-05T03:13:53Z<p>Andykecheng: </p>
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b>A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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</div><br />
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<div class="col_1"><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/ProfileTeam:UCL/Team/Profile2013-10-05T03:13:10Z<p>Andykecheng: </p>
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b>A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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</div><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<div class="col_1"><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
<br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
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</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/f/fd/Andy.png');"><br />
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<br />
<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/1/1b/Robin.png');"><br />
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<br />
<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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</div><br />
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<div class="col_1"><br />
<br />
<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/4/46/Fong_Yi.png');"><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b>A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
<br />
</div><br />
<br />
<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/f/f6/Oran.png');"><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/8/82/John.png');"><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
</div><br />
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
<br />
</div><br />
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<div class="col_1"><br />
<br />
<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/f/fd/Andy.png');"><br />
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<br />
<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
<br />
</div><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b>A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol. </p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book.</p><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.<br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/ProfileTeam:UCL/Team/Profile2013-10-05T03:06:28Z<p>Andykecheng: </p>
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>Sc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
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<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>Sci Natural Sciences..</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
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<p class="minor_title">Tom Johnson</p> <br />
<p class="body_text"><b>MEng Biochemical Enginering.</b> 013/4 Co-president of SynBioSoc. Hobbies include football (FFC) and motorsport. Coordinator of social Thursdays and cultural Tuesdays. Attributions primarily include design and execution of bacterial based laboratory experiments and recording of the lab and general diaries throughout the campaign with responsibility for safety of laboratory experiments.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/4/46/Fong_Yi.png');"><br />
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<br />
<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book. </p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.</p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>MSci Natural Sciences.</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
<br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Team/ProfileTeam:UCL/Team/Profile2013-10-05T03:02:06Z<p>Andykecheng: </p>
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<p class="minor_title">Alex Bates</p> <br />
<p class="body_text"><b>BSc Neuroscience.</b> Being a first year neuroscience student, I was keen to combine my newfound neurobiological knowledge with my nascent interest in synthetic biology. My role is to ground the project in good neuroscience, and conduct wet-lab, bioinformatics and neuroethics work. For me, iGEM is an opportunity to conceive of something truly meaningful and help bring it, quite literally, to life. </p><br />
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<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.<br />
</p><br />
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<p class="minor_title">Weiling (Emily) Yuan</p> <br />
<p class="body_text"><b>MEng Biochemical Engineering.</b> A first year Biochemical Engineering student, interested in synthetic biology, genetic engineering and the human body. I am fascinated by the interdisciplinary nature of iGEM by using a combination of creativity, engineering and science to solve world problems. I am primarily involved in bacterial laboratory work, human practices and company sponsorship.</p><br />
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<p class="minor_title">Fong Yi Khoo</p> <br />
<p class="body_text"><b>BSc Architectural Studies.</b> Now a third-year, I love being thrown in the mad, mad world of art. UCL iGEM has allowed me to be creative but also learn how to blend both art and science. I am working on illustrations, promotional posters, occasional website design and photography.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/4/46/Fong_Yi.png');"><br />
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<p class="minor_title">KhaiCheng Kiew</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> A second-year biochemist interested in medical synthetic biology, gene control and molecular biology. My roles in the project are circuit characterisation, experimental design, mammalian and bacterial lab work, as well as public relations. I enjoy picnics, travelling and have an unfathomable passion for food and 70% ethanol.</p><br />
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<p class="minor_title">Ruxi Comisel</p> <br />
<p class="body_text"><b>BSc Biochemistry.</b> Second year biochemist, interested in synthetic biology as a tool to tackling medical dilemmas. This project answered my preference and resonated with my coming third year research work on Biology of ageing. I’m holding dear topics such as cell regulation and gene expression and my roles in the project are circuit characterisation and bacterial lab work.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/3/37/Ruxi_profile.jpg');"><br />
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<p class="minor_title">Catrin Sohrabi</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> 2nd year Biomedical Scientist. I enjoy applying synthetic biology principles in a practical environment and am excited to contribute and be a part of this years iGEM team. I think our t-shirt is pretty cool. Responsibilities include biobrick construction, work within the bacterial lab and recording of the virtual lab book. </p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Andy Cheng</p> <br />
<p class="body_text"><b>BSc Biomedical Science.</b> As a first year Biomedical Scientist, I am very excited to apply a synthetic biology route towards treating medical disorders. In iGEM, my responsibilities include biobrick construction and coordinating human practices.</p><br />
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<p class="minor_title">Oran Maguire</p> <br />
<p class="body_text"><b>Bsc Human Sciences.</b> Moving into the third year of this degree, I have chosen to focus on neuroscience. I am primarily involved in the design of the website, in providing illustrations and in the redrafting of written work. I also observe and assist in the Bacterial labs.</p><br />
<br />
</div><br />
<br />
<div class="col_1"><br />
<br />
<p class="minor_title">Robin Herd</p> <br />
<p class="body_text"><b>MSci Natural Sciences.</b> As a physical scientist, I will be providing computational support in two forms: modelling and website implementation. I am creating a three-dimensional simulation of the brain which will assist lab work by providing estimates for a number of values, and I am working closely with the team’s artists to create our website.</p><br />
<br />
</div><br />
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<div class="col_2" style="background-image:url('https://static.igem.org/mediawiki/2013/1/1b/Robin.png');"><br />
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<p class="minor_title">StJohn Townsend</p> <br />
<p class="body_text"><b>MSci Genetics.</b> I am a second year geneticist, with a love for SynBio. My roles in the team have been to design the circuit, new parts, and work in both the bacterial and mammalian labs. Hobbies include obscene amounts of primer design and killing vast quantities of HeLa cells.</p><br />
<br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Modeling/BioinformaticsTeam:UCL/Modeling/Bioinformatics2013-10-05T02:58:51Z<p>Andykecheng: </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 />
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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 />
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<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 />
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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 />
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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 />
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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 />
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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 />
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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 />
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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 />
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<th><p class="citation_text">Fig.1 Histogram showing the frequency of gene sets by co-alteration score.</p></th><br />
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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 />
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<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="minor_title">Analysis and Feedback into Circuit</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 <b>BDNF</b> 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>Andykechenghttp://2013.igem.org/Team:UCL/Modeling/BioinformaticsTeam:UCL/Modeling/Bioinformatics2013-10-05T02:57:57Z<p>Andykecheng: </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 />
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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 />
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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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<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 />
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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 />
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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 />
<p class="body_text"><br />
<p class="minor_title">Analysis and Feedback into Circuit</p><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 <b>BDNF</b> 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>Andykechenghttp://2013.igem.org/Team:UCL/Modeling/BioinformaticsTeam:UCL/Modeling/Bioinformatics2013-10-05T02:57:22Z<p>Andykecheng: </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 />
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<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 />
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<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 />
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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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<div class="small_image_left" style="background-image:url('https://static.igem.org/mediawiki/2013/d/d4/Co-alterationScore.gif');height:220px;width:440px"></div><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 />
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<p class="body_text"><br />
‘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 />
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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 />
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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 />
<p class="minor_title">Analysis and Feedback into Circuit</p><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 <b>BDNF</b> 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>Andykechenghttp://2013.igem.org/Team:UCL/Labbook/Week18Team:UCL/Labbook/Week182013-10-05T02:49:49Z<p>Andykecheng: </p>
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<p class="major_title">Lab Weeks</p><br />
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<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 />
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<p class="body_text"><b>Bacterial Labs<b></p><br />
<p class="body_text"><b>Monday 30th September</b></p><br />
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<p class="major_title">October</p><br />
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<p class="body_text"><b>Monday 30th September</b></p><br />
<p class="body_text">Ligated CMV with MMP9 and transformed into Top10 cells. These were subsequently plated on chloarmphenicol plates.<br />
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<p class="body_text"><b>Tuesday 1st October</b></p><br />
<p class="body_text">Inoculated colonies from plates from September 30th.</p><br />
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<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<p class="body_text"> Performed miniprep and generated glycerol stock from incoluation on Oct 1st. </p><br />
<p class="body_text"> Proceeded with analytical digest and gel</p><br />
<p class="body_text"> Transformed CMV+MMP9 plasmid from Sinobiological into Top10 cells</p><br />
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<p class="body_text"><b>Thursday 3nd October</b></p><br />
<p class="body_text"> Religated CMV with MMP9 and continued with transformation into Top10 cells. </p><br />
<p class="body_text"> Inoculated from plates of plasmid from Sinobiological</p><br />
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<p class="body_text"><b>Friday 4th October</b></p><br />
<p class="body_text"> Performed miniprep, generation of glycerol stock on inoculation from Oct 3rd</p><br />
<p class="body_text"> Continued with analytical digest and gel</p><br />
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<b>Mammalian Labs</b><br />
</p><br />
<br />
<p class="body_text"><b>Monday 30th September<b/></p><br />
<br />
<p class="body_text"><b>Tuesday 1st October</b></p><br />
<br />
<p class="body_text"><b>Wednesday 2nd October</b></p><br />
<br />
<p class="body_text"><b>Thursday 3nd October</b></p><br />
<br />
<p class="body_text"><b>Friday 4th October</b></p><br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Practice/DocumentaryTeam:UCL/Practice/Documentary2013-10-05T02:37:07Z<p>Andykecheng: </p>
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<br />
<p class="major_title">EXPLANATORY VIDEO</p><br />
<p class="minor_title">GEM Cells In Plasticine Stop-Motion</p><br />
<p class="body_text"><br />
Communicating ideas in synthetic biology is often difficult, not only because public understanding of the field is limited but because the field is necessarily cross-disciplinary since it tries to apply genetic engineering techniques as new solutions to diverse array of different problems. When making an explanatory video, it is important to be aware of the public perception. For example, genetic engineering is often seen unfavorably with its reputation in genetically modified foodstuffs and fears over eugenics. Neuroscience can cause unease because brain tampering, even for medical purposes, sounds dangerous especially if the method in question seems opaque and amoral to the layman. Our aim in making this short video was to convey our project, in which we fuse these two controversial fields, in a simple and engaging way that does not skimp on the science to make it as translucent and informative as possible. We chose plasticine stop animation because of its simplistic, unassuming, fun feel. <br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<p class="major_title">DOCUMENTARY</p><br />
<p class="minor_title">Interviewing Top Scientists</p><br />
<br />
<p class="body_text"><br />
We are in the process of putting together a documentary on <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics">'neuro-genethics'</a> which will appear on this website later this month. The narrator's script for our documentary can be found here. We also conducted three interviews as a part of our filming process, which have proved invaluable into informing and improving our project work. This documentary explores the views of both science-related professionals and non scientists on the neuroethics and feasibility of neuro-genetic engineering. We prepared a series of questions that targeted the ethical side of brain cell modification for t various purposes, focusing on Alzheimer’s disease, specifically, whether the alteration of native brain cells will prompt a change of ‘self hood’, and how much this kind of new technology can be trusted outside of science. We also examine the economics feasibility of distributing the treatment, looking at resource allocation, the cost of research for Alzheimer's and the cost-benefit of spending on the ageing population. <br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="http://www.iop.kcl.ac.uk/staff/profile/default.aspx?go=10468"target="_blank">Professor John Powell </a></b> - a Professor in Genetics 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.<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/research/personal?upi=PHAGG98" target="_blank">Professor Patrick Haggard</a></b> - A prominent figure in neuroethical debate, Patrick Haggard is is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, University College London (UCL). His interests lie in voluntary action, and so the question of whether or not we have 'free will', as well as how the brain represents an individual's body within themselves, and so the question of selfhood. <br />
</p><br />
<p class="body_text"><br />
Professor Patrick Haggard, who specializes in the neurophysiology of free will, met us on the 26th of July to discuss our project. During this meeting, we covered areas such as the uniqueness of our project and the ethical issues revolving around preventing degeneration and compromising identity.<br />
He emphasized how we would be changing the person as we would be changing the bits and pieces that are central to the person’s identity, the brain. We are working with biological circuits that are closely related to the humanity of the individual. It is completely different to genetic engineering hair or the kidneys. Click <a href="https://static.igem.org/mediawiki/2013/4/46/Haggard.mp3" target="_blank">here</a> to listen to the audio sample from the documentary.<br />
</p><br />
<p class="body_text"><br />
Furthermore, he points out that our project targets on dementia rather than behavioral and movement disorders as do most current research does. With dementia, the goal is to restore the capacity to remember and process information. The outcome is intrinsically subjective. This is contrasted with treating movement disorders such as Parkinson’s with electrical stimulation. He warns us about the risks in our intervention. The main question that needs to be addressed is how much of an individual’s identity or memory would be comprised. He asks us whether we would be able to show people our treatment would not modify memories or interfere with central brain circuits that give us our identity. <br />
</p><br />
<p class="body_text"><br />
Next, we discussed whether there would be any additional risks with using genetic engineering in the brain. Both these fields are of high risk, but Professor Haggard believes there is no additional risk from this overlap. He believes the risks with any intervening treatments in the brain are universal, such as pharmacological ones. Drugs, machinery or genetically modified cells all disturb the brain, therefore there should not be any additional concern when compared with other more conventional treatments.<br />
</p><br />
<p class="body_text"><br />
Many people have ethical concerns about technology that prolongs people’s lives indefinitely, Professor Haggard is definitely one of them. He says that degeneration is a natural process and people ought to accept it. The ethics of moving towards ‘immortality’ is new and underdeveloped as modern medicine is still far from achieving ‘immortality’. He admits that he tolerates the natural degeneration of his body but he refuses to allow his mind to degenerate. The mind is so closely linked with the capacity to enhance the happiness of the people around us and ourselves. It is also the basis of our personality. The thought of losing one self and one’s memories is horrifying and tragic. Professor Haggard states that if modern medicine is truly moving towards granting immortality, it is correct how we chose to target a neurodegenerative disease. <br />
<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/iris/browse/profile?upi=SLHAR52" target="_blank">Professor Stephen Hart</a></b> - Stephen Hart works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We contacting him for interview because we wanted to discuss methods of gene delivery to the brain that could be incorporated into the clinical theory behind our genetic circuit. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using <a href="https://2013.igem.org/Team:UCL/Project/Chemotaxis">lipid-peptide nanocomplexes</a>. 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. It is a great example of how our synthetic neurobiological treatment could be brought to the clinic, and selectively target microglia and could be used to develop microglia as a chassis for gene and drug delivery.<br />
</p><br />
<br />
<div class="gap"></div><br />
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<br />
<p class="major_title">EXPLANATORY VIDEO</p><br />
<p class="minor_title">GEM Cells In Plasticine Stop-Motion</p><br />
<p class="body_text"><br />
Communicating ideas in synthetic biology is often difficult, not only because public understanding of the field is limited but because the field is necessarily cross-disciplinary since it tries to apply genetic engineering techniques as new solutions to diverse array of different problems. When making an explanatory video, it is important to be aware of the public perception. For example, genetic engineering is often seen unfavorably with its reputation in genetically modified foodstuffs and fears over eugenics. Neuroscience can cause unease because brain tampering, even for medical purposes, sounds dangerous especially if the method in question seems opaque and amoral to the layman. Our aim in making this short video was to convey our project, in which we fuse these two controversial fields, in a simple and engaging way that does not skimp on the science to make it as translucent and informative as possible. We chose plasticine stop animation because of its simplistic, unassuming, fun feel. <br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<p class="major_title">DOCUMENTARY</p><br />
<p class="minor_title">Interviewing Top Scientists</p><br />
<br />
<p class="body_text"><br />
We are in the process of putting together a documentary on <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics">'neuro-genethics'</a> which will appear on this website later this month. The narrator's script for our documentary can be found here. We also conducted three interviews as a part of our filming process, which have proved invaluable into informing and improving our project work. This documentary explores the views of both science-related professionals and non scientists on the neuroethics and feasibility of neuro-genetic engineering. We prepared a series of questions that targeted the ethical side of brain cell modification for t various purposes, focusing on Alzheimer’s disease, specifically, whether the alteration of native brain cells will prompt a change of ‘self hood’, and how much this kind of new technology can be trusted outside of science. We also examine the economics feasibility of distributing the treatment, looking at resource allocation, the cost of research for Alzheimer's and the cost-benefit of spending on the ageing population. <br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="http://www.iop.kcl.ac.uk/staff/profile/default.aspx?go=10468"target="_blank">Professor John Powell </a></b> - a Professor in Genetics 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.<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/research/personal?upi=PHAGG98" target="_blank">Professor Patrick Haggard</a></b> - A prominent figure in neuroethical debate, Patrick Haggard is is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, University College London (UCL). His interests lie in voluntary action, and so the question of whether or not we have 'free will', as well as how the brain represents an individual's body within themselves, and so the question of selfhood. <br />
</p><br />
<p class="body_text"><br />
Professor Patrick Haggard, who specializes in the neurophysiology of free will, met us on the 26th of July to discuss our project. During this meeting, we covered areas such as the uniqueness of our project and the ethical issues revolving around preventing degeneration and compromising identity.<br />
He emphasized how we would be changing the person as we would be changing the bits and pieces that are central to the person’s identity, the brain. We are working with biological circuits that are closely related to the humanity of the individual. It is completely different to genetic engineering hair or the kidneys. Click <a href="https://static.igem.org/mediawiki/2013/4/46/Haggard.mp3" target="_blank">here</a>to listen to the audio sample from the documentary.<br />
</p><br />
<p class="body_text"><br />
Furthermore, he points out that our project targets on dementia rather than behavioral and movement disorders as do most current research does. With dementia, the goal is to restore the capacity to remember and process information. The outcome is intrinsically subjective. This is contrasted with treating movement disorders such as Parkinson’s with electrical stimulation. He warns us about the risks in our intervention. The main question that needs to be addressed is how much of an individual’s identity or memory would be comprised. He asks us whether we would be able to show people our treatment would not modify memories or interfere with central brain circuits that give us our identity. <br />
</p><br />
<p class="body_text"><br />
Next, we discussed whether there would be any additional risks with using genetic engineering in the brain. Both these fields are of high risk, but Professor Haggard believes there is no additional risk from this overlap. He believes the risks with any intervening treatments in the brain are universal, such as pharmacological ones. Drugs, machinery or genetically modified cells all disturb the brain, therefore there should not be any additional concern when compared with other more conventional treatments.<br />
</p><br />
<p class="body_text"><br />
Many people have ethical concerns about technology that prolongs people’s lives indefinitely, Professor Haggard is definitely one of them. He says that degeneration is a natural process and people ought to accept it. The ethics of moving towards ‘immortality’ is new and underdeveloped as modern medicine is still far from achieving ‘immortality’. He admits that he tolerates the natural degeneration of his body but he refuses to allow his mind to degenerate. The mind is so closely linked with the capacity to enhance the happiness of the people around us and ourselves. It is also the basis of our personality. The thought of losing one self and one’s memories is horrifying and tragic. Professor Haggard states that if modern medicine is truly moving towards granting immortality, it is correct how we chose to target a neurodegenerative disease. <br />
<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/iris/browse/profile?upi=SLHAR52" target="_blank">Professor Stephen Hart</a></b> - Stephen Hart works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We contacting him for interview because we wanted to discuss methods of gene delivery to the brain that could be incorporated into the clinical theory behind our genetic circuit. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using <a href="https://2013.igem.org/Team:UCL/Project/Chemotaxis">lipid-peptide nanocomplexes</a>. 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. It is a great example of how our synthetic neurobiological treatment could be brought to the clinic, and selectively target microglia and could be used to develop microglia as a chassis for gene and drug delivery.<br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_medium" style="background-image:url('https://static.igem.org/mediawiki/2013/c/c0/Plasticine_banner_UCL.png');height:400px;width:1000px"></div><br />
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<br />
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</p><br />
<p class="body_text"><br />
<b><a href="http://c-lab.co.uk/events/speed-debate-on-synthetic-biology-and-neuro-ethics.html" target="_blank">Speed Debate on Synthetic Biology and Neuro-ethics</a></b><br />
</p><br />
</div><br />
<br />
<p class="minor_title">Science Prophet</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7a/Science-prophet.jpg');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://www.scienceprophet.com/Articles/Marvels%20and%20Morals/" target="_blank">Marvels And Morals</a></b><br />
<br />
<br />
<p class="minor_title">London Futurists</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/f/fc/Futurists.png');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://www.meetup.com/London-Futurists/messages/boards/thread/3638333" target="_blank">Open Debate on the Ethics of Neuro-Engineering and synthetic biology</a></b><br />
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<p class="body_text"><br />
<b><a href="http://c-lab.co.uk/events/speed-debate-on-synthetic-biology-and-neuro-ethics.html" target="_blank">Speed Debate on Synthetic Biology and Neuro-ethics</a></b><br />
</p><br />
</div><br />
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<p class="minor_title">Science Prophet</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7a/Science-prophet.jpg');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://www.scienceprophet.com/Articles/Marvels%20and%20Morals/" target="_blank">Marvels And Morals</a></b><br />
<br />
<br />
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<p class="major_title">Media Watch</p><br />
<br />
<p class="minor_title">C-LAB</p><br />
<br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/1/15/Logo_68x63.gif');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://c-lab.co.uk/events/speed-debate-on-synthetic-biology-and-neuro-ethics.html" target="_blank">Speed Debate on Synthetic Biology and Neuro-ethics</a></b><br />
</p><br />
</div><br />
<br />
<p class="minor_title">London Futurists</p><br />
<br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/f/fc/Futurists.png');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://www.meetup.com/London-Futurists/messages/boards/thread/36383332">London Futurists</a></b><br />
</p><br />
</div><br />
<br />
<p class="minor_title">Science Prophet</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7a/Science-prophet.jpg');height:79px;width:89px"></div><br />
</p><br />
<p class="body_text"><br />
<b><a href="http://www.scienceprophet.com/Articles/Marvels%20and%20Morals/" target="_blank">Marvels And Morals</a></b><br />
<br />
<br />
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</html></div>Andykechenghttp://2013.igem.org/File:Futurists.pngFile:Futurists.png2013-10-05T02:27:42Z<p>Andykecheng: </p>
<hr />
<div></div>Andykechenghttp://2013.igem.org/File:Haggard.mp3File:Haggard.mp32013-10-05T02:26:42Z<p>Andykecheng: </p>
<hr />
<div></div>Andykechenghttp://2013.igem.org/Team:UCL/Practice/DocumentaryTeam:UCL/Practice/Documentary2013-10-05T02:16:47Z<p>Andykecheng: </p>
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<br />
<p class="major_title">EXPLANATORY VIDEO</p><br />
<p class="minor_title">GEM Cells In Plasticine Stop-Motion</p><br />
<p class="body_text"><br />
Communicating ideas in synthetic biology is often difficult, not only because public understanding of the field is limited but because the field is necessarily cross-disciplinary since it tries to apply genetic engineering techniques as new solutions to diverse array of different problems. When making an explanatory video, it is important to be aware of the public perception. For example, genetic engineering is often seen unfavorably with its reputation in genetically modified foodstuffs and fears over eugenics. Neuroscience can cause unease because brain tampering, even for medical purposes, sounds dangerous especially if the method in question seems opaque and amoral to the layman. Our aim in making this short video was to convey our project, in which we fuse these two controversial fields, in a simple and engaging way that does not skimp on the science to make it as translucent and informative as possible. We chose plasticine stop animation because of its simplistic, unassuming, fun feel. <br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<p class="major_title">DOCUMENTARY</p><br />
<p class="minor_title">Interviewing Top Scientists</p><br />
<br />
<p class="body_text"><br />
We are in the process of putting together a documentary on <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics">'neuro-genethics'</a> which will appear on this website later this month. The narrator's script for our documentary can be found here. We also conducted three interviews as a part of our filming process, which have proved invaluable into informing and improving our project work. This documentary explores the views of both science-related professionals and non scientists on the neuroethics and feasibility of neuro-genetic engineering. We prepared a series of questions that targeted the ethical side of brain cell modification for t various purposes, focusing on Alzheimer’s disease, specifically, whether the alteration of native brain cells will prompt a change of ‘self hood’, and how much this kind of new technology can be trusted outside of science. We also examine the economics feasibility of distributing the treatment, looking at resource allocation, the cost of research for Alzheimer's and the cost-benefit of spending on the ageing population. <br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="http://www.iop.kcl.ac.uk/staff/profile/default.aspx?go=10468"target="_blank">Professor John Powell </a></b> - a Professor in Genetics 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.<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/research/personal?upi=PHAGG98" target="_blank">Professor Patrick Haggard</a></b> - A prominent figure in neuroethical debate, Patrick Haggard is is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, University College London (UCL). His interests lie in voluntary action, and so the question of whether or not we have 'free will', as well as how the brain represents an individual's body within themselves, and so the question of selfhood. <br />
</p><br />
<p class="body_text"><br />
Professor Patrick Haggard, who specializes in the neurophysiology of free will, met us on the 26th of July to discuss our project. During this meeting, we covered areas such as the uniqueness of our project and the ethical issues revolving around preventing degeneration and compromising identity.<br />
He emphasized how we would be changing the person as we would be changing the bits and pieces that are central to the person’s identity, the brain. We are working with biological circuits that are closely related to the humanity of the individual. It is completely different to genetic engineering hair or the kidneys. <br />
</p><br />
<p class="body_text"><br />
Furthermore, he points out that our project targets on dementia rather than behavioral and movement disorders as do most current research does. With dementia, the goal is to restore the capacity to remember and process information. The outcome is intrinsically subjective. This is contrasted with treating movement disorders such as Parkinson’s with electrical stimulation. He warns us about the risks in our intervention. The main question that needs to be addressed is how much of an individual’s identity or memory would be comprised. He asks us whether we would be able to show people our treatment would not modify memories or interfere with central brain circuits that give us our identity. <br />
</p><br />
<p class="body_text"><br />
Next, we discussed whether there would be any additional risks with using genetic engineering in the brain. Both these fields are of high risk, but Professor Haggard believes there is no additional risk from this overlap. He believes the risks with any intervening treatments in the brain are universal, such as pharmacological ones. Drugs, machinery or genetically modified cells all disturb the brain, therefore there should not be any additional concern when compared with other more conventional treatments.<br />
</p><br />
<p class="body_text"><br />
Many people have ethical concerns about technology that prolongs people’s lives indefinitely, Professor Haggard is definitely one of them. He says that degeneration is a natural process and people ought to accept it. The ethics of moving towards ‘immortality’ is new and underdeveloped as modern medicine is still far from achieving ‘immortality’. He admits that he tolerates the natural degeneration of his body but he refuses to allow his mind to degenerate. The mind is so closely linked with the capacity to enhance the happiness of the people around us and ourselves. It is also the basis of our personality. The thought of losing one self and one’s memories is horrifying and tragic. Professor Haggard states that if modern medicine is truly moving towards granting immortality, it is correct how we chose to target a neurodegenerative disease. <br />
<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/iris/browse/profile?upi=SLHAR52" target="_blank">Professor Stephen Hart</a></b> - Stephen Hart works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We contacting him for interview because we wanted to discuss methods of gene delivery to the brain that could be incorporated into the clinical theory behind our genetic circuit. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using <a href="https://2013.igem.org/Team:UCL/Project/Chemotaxis">lipid-peptide nanocomplexes</a>. 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. It is a great example of how our synthetic neurobiological treatment could be brought to the clinic, and selectively target microglia and could be used to develop microglia as a chassis for gene and drug delivery.<br />
</p><br />
<br />
<div class="gap"></div><br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Practice/DocumentaryTeam:UCL/Practice/Documentary2013-10-05T02:15:10Z<p>Andykecheng: </p>
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<br />
<p class="major_title">EXPLANATORY VIDEO</p><br />
<p class="minor_title">GEM Cells In Plasticine Stop-Motion</p><br />
<p class="body_text"><br />
Communicating ideas in synthetic biology is often difficult, not only because public understanding of the field is limited but because the field is necessarily cross-disciplinary since it tries to apply genetic engineering techniques as new solutions to diverse array of different problems. When making an explanatory video, it is important to be aware of the public perception. For example, genetic engineering is often seen unfavorably with its reputation in genetically modified foodstuffs and fears over eugenics. Neuroscience can cause unease because brain tampering, even for medical purposes, sounds dangerous especially if the method in question seems opaque and amoral to the layman. Our aim in making this short video was to convey our project, in which we fuse these two controversial fields, in a simple and engaging way that does not skimp on the science to make it as translucent and informative as possible. We chose plasticine stop animation because of its simplistic, unassuming, fun feel. <br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<p class="major_title">DOCUMENTARY</p><br />
<p class="minor_title">Interviewing Top Scientists</p><br />
<br />
<p class="body_text"><br />
We are in the process of putting together a documentary on <a href="https://2013.igem.org/Team:UCL/Practice/Neuroethics">'neuro-genethics'</a> which will appear on this website later this month. The narrator's script for our documentary can be found here. We also conducted three interviews as a part of our filming process, which have proved invaluable into informing and improving our project work. This documentary explores the views of both science-related professionals and non scientists on the neuroethics and feasibility of neuro-genetic engineering. We prepared a series of questions that targeted the ethical side of brain cell modification for t various purposes, focusing on Alzheimer’s disease, specifically, whether the alteration of native brain cells will prompt a change of ‘self hood’, and how much this kind of new technology can be trusted outside of science. We also examine the economics feasibility of distributing the treatment, looking at resource allocation, the cost of research for Alzheimer's and the cost-benefit of spending on the ageing population. <br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="http://www.iop.kcl.ac.uk/staff/profile/default.aspx?go=10468"target="_blank">Professor John Powell </a></b> - a Professor in Genetics 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.<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/research/personal?upi=PHAGG98" target="_blank">Professor Patrick Haggard</a></b> - A prominent figure in neuroethical debate, Patrick Haggard is is a neuroscientist at the Institute of Cognitive Neuroscience and the Department of Psychology, University College London (UCL). His interests lie in voluntary action, and so the question of whether or not we have 'free will', as well as how the brain represents an individual's body within themselves, and so the question of selfhood. <br />
<br />
Professor Patrick Haggard, who specializes in the neurophysiology of free will, met us on the 26th of July to discuss our project. During this meeting, we covered areas such as the uniqueness of our project and the ethical issues revolving around preventing degeneration and compromising identity.<br />
He emphasized how we would be changing the person as we would be changing the bits and pieces that are central to the person’s identity, the brain. We are working with biological circuits that are closely related to the humanity of the individual. It is completely different to genetic engineering hair or the kidneys. <br />
Furthermore, he points out that our project targets on dementia rather than behavioral and movement disorders as do most current research does. With dementia, the goal is to restore the capacity to remember and process information. The outcome is intrinsically subjective. This is contrasted with treating movement disorders such as Parkinson’s with electrical stimulation. He warns us about the risks in our intervention. The main question that needs to be addressed is how much of an individual’s identity or memory would be comprised. He asks us whether we would be able to show people our treatment would not modify memories or interfere with central brain circuits that give us our identity. <br />
Next, we discussed whether there would be any additional risks with using genetic engineering in the brain. Both these fields are of high risk, but Professor Haggard believes there is no additional risk from this overlap. He believes the risks with any intervening treatments in the brain are universal, such as pharmacological ones. Drugs, machinery or genetically modified cells all disturb the brain, therefore there should not be any additional concern when compared with other more conventional treatments.<br />
Many people have ethical concerns about technology that prolongs people’s lives indefinitely, Professor Haggard is definitely one of them. He says that degeneration is a natural process and people ought to accept it. The ethics of moving towards ‘immortality’ is new and underdeveloped as modern medicine is still far from achieving ‘immortality’. He admits that he tolerates the natural degeneration of his body but he refuses to allow his mind to degenerate. The mind is so closely linked with the capacity to enhance the happiness of the people around us and ourselves. It is also the basis of our personality. The thought of losing one self and one’s memories is horrifying and tragic. Professor Haggard states that if modern medicine is truly moving towards granting immortality, it is correct how we chose to target a neurodegenerative disease. <br />
<br />
</p><br />
<br />
<p class="body_text"><br />
<b><a href="https://iris.ucl.ac.uk/iris/browse/profile?upi=SLHAR52" target="_blank">Professor Stephen Hart</a></b> - Stephen Hart works on gene therapy at Wolfson Centre for Gene Therapy of Childhood Disease, UCL. We contacting him for interview because we wanted to discuss methods of gene delivery to the brain that could be incorporated into the clinical theory behind our genetic circuit. By pure serendipity we found that he and his research team had developed a method of transfecting microglia in vivo using <a href="https://2013.igem.org/Team:UCL/Project/Chemotaxis">lipid-peptide nanocomplexes</a>. 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. It is a great example of how our synthetic neurobiological treatment could be brought to the clinic, and selectively target microglia and could be used to develop microglia as a chassis for gene and drug delivery.<br />
</p><br />
<br />
<div class="gap"></div><br />
<br />
<div class="row_medium" style="background-image:url('https://static.igem.org/mediawiki/2013/c/c0/Plasticine_banner_UCL.png');height:400px;width:1000px"></div><br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:29:26Z<p>Andykecheng: </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 />
</p><br />
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<p class="major_title">Bacterial Lab Protocols</p><br />
<div class="gap"></div><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 />
<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 />
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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 />
</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 />
<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: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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<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 />
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<p class="major_title">Bacterial Lab Protocols</p><br />
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<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 />
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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 />
</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 />
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<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 />
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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 />
</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 />
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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 />
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<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 />
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<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><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 />
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<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 />
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<div class="gap"><br />
</div><br />
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<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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<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="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 />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:22:13Z<p>Andykecheng: </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><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>50ul Temperature</th><br />
<th>Final 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>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 />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</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="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="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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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:16:51Z<p>Andykecheng: </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 />
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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 />
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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><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 />
<br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</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="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>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:12:45Z<p>Andykecheng: </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"><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><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 500mL Duran bottle insert:<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>RO H2O</td><br />
<td>500mL</td><br />
</tr><br />
<tr><br />
</table><br />
</div><br />
<div class="description"><br />
<p class="minor_title">Ligation</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="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 />
<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: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 />
<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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<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><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">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="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 />
<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: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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<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><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 />
<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="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> <br />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:07:03Z<p>Andykecheng: Undo revision 309676 by Andykecheng (talk)</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 />
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<div class="gap"><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"><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="gap"></div><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 />
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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="gap"></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 />
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<div class="gap"></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><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 />
<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="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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<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 />
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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>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:06:32Z<p>Andykecheng: </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"><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 />
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<div class="gap"></div><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 />
<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><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 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="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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<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="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 />
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</html></div>Andykechenghttp://2013.igem.org/Team:UCL/Project/ProtocolsTeam:UCL/Project/Protocols2013-10-05T00:06:00Z<p>Andykecheng: </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 />
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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 />
<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 />
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<div class="gap"></div><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 />
</div><br />
</div><br />
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<div class="gap"></div><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 />
<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><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 />
<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="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>Andykechenghttp://2013.igem.org/Team:UCL/Labbook/Week16Team:UCL/Labbook/Week162013-10-04T23:55:47Z<p>Andykecheng: </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 J632014 with E and 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>J632014 DNA</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 />
Miniprep 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 37OC. 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 miniprep 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 Ecor1 (THIS IS INCORRECT)</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 transformation 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 />
Nanodrops Readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90<br />
</p><br />
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<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 J632014 with E and 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>J632014 DNA</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 />
Miniprep 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 37OC. 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 miniprep 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 Ecor1 (THIS IS INCORRECT)</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 />
<br />
<p class="body_text"><br />
Re-Inocubations from glycerol stocks from both batches of transformation 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 />
Nanodrops Readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90<br />
</p><br />
<br />
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<p class="major_title">Lab Weeks</p><br />
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<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 J632014 with E and 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>J632014 DNA</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 <b>INSERT IMAGE OF GEL HERE PLS</B><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 />
Miniprep 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 37OC. 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 miniprep 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 Ecor1 (THIS IS INCORRECT)</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 />
<br />
<p class="body_text"><br />
Re-Inocubations from glycerol stocks from both batches of transformation 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 />
Nanodrops Readings (after maxi-prep) of Zec sample 1, ng/ul = 40.7, 260/280 = 1.90<br />
</p><br />
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<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 />
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<p class="minor_title">Week 15</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 9th September<b><br />
</p><br />
<p class="body_text"><br />
Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.<br />
</p><br />
<p class="body_text"><br />
Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.<br />
</p><br />
<p class="body_text"><br />
Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.<br />
</p><br />
<p class="body_text"><br />
LIGATION 3:<br />
</p><br />
<p class="body_text"><br />
pSB1C3 digested and purified, concentration = 25 ng/ul <br />
<br />
</p><br />
<p class="body_text"><br />
zeo digested and purified conc = 77 ng/ul<br />
</p><br />
<p class="body_text"><br />
CMV digested and purified conc = 25 ng/ul<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (77 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [z1]</th><br />
<th>6 to 1 (mass ratio) [z2]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>6</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>2</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Cmv (25 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [c3]</th><br />
<th>6 to 1 (mass ratio) [c4]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>2</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>5 ctrl check no circular backbone</th><br />
<th>6 ctrl check digestion process</th><br />
<th>7 ctrl (uncut backbone)</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>7</td><br />
<td>18</td><br />
<td>18</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>3</td><br />
<td>3</td><br />
<td>3 of uncut backbone</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.<br />
</p><br />
<br />
<p class="body_text"><br />
Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated overnight and then minipreped.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 10th September<b><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the re-transformation of ligations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony counts</th><br />
</tr><br />
<tr><br />
<td>2z from 31/08</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>5z from 4/09</td><br />
<td>150+</td><br />
</tr><br />
<tr><br />
<td>6z from 4/09</td><br />
<td>80+</td><br />
</tr><br />
<tr><br />
<td>7z from 4/09</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>8z from 4/09</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>9z - ctrl from 4/09</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>11z - ctrl from 4/09</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock. <br />
</p><br />
<p class="body_text"><br />
Colony count - 90% - 100+ and 10% 80 colonies.<br />
</p><br />
<br />
<p class="body_text"><br />
Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.<br />
</p><br />
<p class="body_text"><br />
The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.<br />
</p><br />
<p class="body_text"><br />
The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes.<br />
The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.<br />
</p><br />
<p class="body_text"><br />
Building up the competent cell stock using the home-made competent cells<br />
</p><br />
<p class="body_text"><br />
Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate number</th><br />
<th>Drug</th><br />
<th>Volume competent cells (ul)</th><br />
<th>Colony count</th><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
<tr><br />
<td>II</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>III</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>IV</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>V</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>VI</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>VII</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>VIII</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>IX</td><br />
<td>1000 X CMP</td><br />
<td>100 from T vial</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>X</td><br />
<td>N/A</td><br />
<td>100 from T vial</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.<br />
</p><br />
<p class="body_text"><br />
New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.<br />
<br />
</p><br />
<p class="body_text"><br />
Transformation number 3:<br />
</p><br />
<p class="body_text"><br />
We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.<br />
</p><br />
<p class="body_text"><br />
The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.<br />
</p><br />
<p class="body_text"><br />
After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.<br />
</p><br />
<p class="body_text"><br />
On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 11th Sepetmber</b><br />
</p><br />
<br />
<p class="body_text"><br />
Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. <br />
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.<br />
</p><br />
<br />
<p class="body_text"><br />
Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Falcon Tube no.</th><br />
<th>Content: Plate & Colony</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>2z.1</td><br />
<td>54.0</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>2z.2</td><br />
<td>28.3</td><br />
<td>2.21</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>2z.3</td><br />
<td>25.6</td><br />
<td>3.03</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>2z.4</td><br />
<td>20.3</td><br />
<td>3.16</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>2z.5</td><br />
<td>13.4</td><br />
<td>3.08</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>2z.6</td><br />
<td>15.7</td><br />
<td>2.61</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>2z.7</td><br />
<td>8.9</td><br />
<td>14.17</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>2z.8</td><br />
<td>14.8</td><br />
<td>4.08</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>5z.1</td><br />
<td>64.9</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>5z.2</td><br />
<td>36.5</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>5z.3</td><br />
<td>16.6</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>5z.4</td><br />
<td>32.1</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>5z.5</td><br />
<td>23.6</td><br />
<td>2.50</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>5z.6</td><br />
<td>53.3</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>5z.7</td><br />
<td>33.7</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>16</td><br />
<td>5z.8</td><br />
<td>50.1</td><br />
<td>2.06</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>6z.1</td><br />
<td>74.8</td><br />
<td>1.71</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>6z.2</td><br />
<td>32.6</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>19</td><br />
<td>6z.3</td><br />
<td>26.0</td><br />
<td>2.25</td><br />
</tr><br />
<tr><br />
<td>20</td><br />
<td>6z.4</td><br />
<td>42.5</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>6z.5</td><br />
<td>30.8</td><br />
<td>2.02</td><br />
</tr><br />
<tr><br />
<td>22</td><br />
<td>6z.6</td><br />
<td>27.3</td><br />
<td>2.23</td><br />
</tr><br />
<tr><br />
<td>23</td><br />
<td>6z.7</td><br />
<td>38.0</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>24</td><br />
<td>6z.8</td><br />
<td>43.5</td><br />
<td>2.10</td><br />
</tr><br />
<tr><br />
<td>25</td><br />
<td>7z.1</td><br />
<td>62.7</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>7z.2</td><br />
<td>31.7</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>27</td><br />
<td>7z.3</td><br />
<td>26.4</td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>7z.4</td><br />
<td>21.4</td><br />
<td>2.12</td><br />
</tr><br />
<tr><br />
<td>29</td><br />
<td>7z.5</td><br />
<td>13.1</td><br />
<td>2.28</td><br />
</tr><br />
<tr><br />
<td>30</td><br />
<td>7z.6</td><br />
<td>27.2</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>31</td><br />
<td>7z.7</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>32</td><br />
<td>7z.8</td><br />
<td>33.0</td><br />
<td>2.28</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the transformed cells with the ligation prepared on the 9/09<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony Count</th><br />
</tr><br />
<tr><br />
<td>Z1</td><br />
<td>12</td><br />
</tr><br />
<tr><br />
<td>Z2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>C4</td><br />
<td>1 (+small colonies)</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>17</td><br />
</tr><br />
<tr><br />
<td>pSecTag2A</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).<br />
</p><br />
<p class="body_text"><br />
Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).<br />
</p><br />
<p class="body_text"><br />
Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands:<br />
- For zeocin 2 bands: one of 1037 bp and another of 783 bp<br />
- For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp<br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/4/44/Herpaderpa.png');height:527px;width:700px"></div><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Item</th><br />
<th>ul</th><br />
</tr><br />
<tr><br />
<td>DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul)</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>3</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 />
The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul).<br />
These cuts were run on a gel, next to uncuts.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 12th September<b><br />
</p><br />
<p class="body_text"><br />
Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>14</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>30</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.<br />
</p><br />
<p class="body_text"><br />
Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.<br />
</p><br />
<br />
<p class="body_text"><br />
Ligation 4<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Ligations</th><br />
<th>AA1</th><br />
<th>BB1</th><br />
<th>CC1</th><br />
</tr><br />
<tr><br />
<td>EP cut, purified pAZEC fragment (53ng/ul)</td><br />
<td>2</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>*EPD cut pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>T4 DNA ligase buffer</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>**Vol. of dilute T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>RO Water</td><br />
<td>4</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Cell Counts</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;<br />
</p><br />
<p class="body_text"><br />
** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex.<br />
These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.<br />
</p><br />
<p class="body_text"><br />
<u>Transformation 4</u><br />
</p><br />
<p class="body_text"><br />
Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.<br />
3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 13th September</b><br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin using <a href="https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction" target="_blank"> Taq PCR kit New England Biolabs</a> in 50 ul reaction volume.<br />
</p><br />
<p class="body_text"><br />
Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09<br />
</p><br />
<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>Z1.1</td><br />
<td>24.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>Z1.2</td><br />
<td>35.4</td><br />
<td>1.98</td><br />
</tr><br />
<tr><br />
<td>Z1.3</td><br />
<td>34.2</td><br />
<td>1.93</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Accomplished tasks asked by Darren:<br />
</p><br />
<p class="body_text"><br />
- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.<br />
</p><br />
<p class="body_text"><br />
- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.<br />
</p><br />
<p class="body_text"><br />
- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).<br />
</p><br />
<p class="body_text"><br />
- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.<br />
</p><br />
<p class="body_text"><br />
Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band). <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7b/Sep_13_UCLiGEM2013.png');height:554px;width:650px"></div><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>E&P digest (ul)</th><br />
<th>BscR1 digest (ul)</th><br />
<th>Stu1 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>EcoR1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BscR1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</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>RO H2O</td><br />
<td>1.5</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 />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 14th September</b><br />
</p><br />
<p class="body_text"><br />
Colony counts of the transformation prepared on 12th and 13th of September.<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>AA1 5xcmp</th><br />
<th>AA1 2xcmp</th><br />
<th>AA1 1xcmp</th><br />
<th>BB1 5xcmp</th><br />
<th>BB1 2xcmp</th><br />
<th>BB1 1xcmp</th><br />
<th>CC1 5xcmp</th><br />
<th>CC1 2xcmp</th><br />
<th>CC1 1xcmp</th><br />
</tr><br />
<tr><br />
<td>12th Sep</td><br />
<td>1</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>28</td><br />
<td>-</td><br />
<td>0</td><br />
<td>0</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>13th Sep</td><br />
<td>0</td><br />
<td>-</td><br />
<td>30</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>0</td><br />
<td>-</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of these minipreps – data from the next day<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>AA1 1xcmp colony number</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>26.8</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>24.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>35.1</td><br />
<td>1.90</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>28.2</td><br />
<td>1.95</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>30.5</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>28.7</td><br />
<td>1.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>35.1</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>40.3</td><br />
<td>1.88</td><br />
</tr><br />
</table><br />
<p><br />
<br />
<p class="body_text"><br />
The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.<br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.<br />
</p><br />
<p class="body_text"><br />
A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 15th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.<br />
</p><br />
<p class="body_text"><br />
From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.<br />
</p><br />
<p class="body_text"><br />
Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Volume (ul)</th><br />
<th>MMP9 4bb</th><br />
<th>MMP9 4bb</th><br />
<th>Control (no backbone)</th><br />
<th>Control (no insert)</th><br />
<th>MMP9 3</th><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>5</td><br />
<td>7.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (from K812014)</td><br />
<td>0</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (hs)</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 />
<td>10</td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Insert - MMP9</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>0</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Vial Labels</td><br />
<td>1</td><br />
<td>2</td><br />
<td>3</td><br />
<td>4</td><br />
<td>5</td><br />
</tr><br />
</table><br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Tuesday 10th September</b><br />
</p><br />
<p class="body_text"><br />
Threw away 12 over-confluent ( aprox. 150%) plates (P15).<br />
5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.<br />
</p><br />
<p class="body_text"><br />
Added FBS and PS intp DMEM media to make new solution.<br />
</p><br />
</div><br />
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</html></div>Andykechenghttp://2013.igem.org/File:Herpaderpa.pngFile:Herpaderpa.png2013-10-04T23:13:57Z<p>Andykecheng: </p>
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<div></div>Andykechenghttp://2013.igem.org/Team:UCL/Labbook/Week15Team:UCL/Labbook/Week152013-10-04T23:04:15Z<p>Andykecheng: </p>
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<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 15</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 9th September<b><br />
</p><br />
<p class="body_text"><br />
Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.<br />
</p><br />
<p class="body_text"><br />
Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.<br />
</p><br />
<p class="body_text"><br />
Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.<br />
</p><br />
<p class="body_text"><br />
LIGATION 3:<br />
</p><br />
<p class="body_text"><br />
pSB1C3 digested and purified, concentration = 25 ng/ul <br />
<br />
</p><br />
<p class="body_text"><br />
zeo digested and purified conc = 77 ng/ul<br />
</p><br />
<p class="body_text"><br />
CMV digested and purified conc = 25 ng/ul<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (77 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [z1]</th><br />
<th>6 to 1 (mass ratio) [z2]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>6</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>2</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Cmv (25 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [c3]</th><br />
<th>6 to 1 (mass ratio) [c4]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>2</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>5 ctrl check no circular backbone</th><br />
<th>6 ctrl check digestion process</th><br />
<th>7 ctrl (uncut backbone)</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>7</td><br />
<td>18</td><br />
<td>18</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>3</td><br />
<td>3</td><br />
<td>3 of uncut backbone</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.<br />
</p><br />
<br />
<p class="body_text"><br />
Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated overnight and then minipreped.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 10th September<b><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the re-transformation of ligations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony counts</th><br />
</tr><br />
<tr><br />
<td>2z from 31/08</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>5z from 4/09</td><br />
<td>150+</td><br />
</tr><br />
<tr><br />
<td>6z from 4/09</td><br />
<td>80+</td><br />
</tr><br />
<tr><br />
<td>7z from 4/09</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>8z from 4/09</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>9z - ctrl from 4/09</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>11z - ctrl from 4/09</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock. <br />
</p><br />
<p class="body_text"><br />
Colony count - 90% - 100+ and 10% 80 colonies.<br />
</p><br />
<br />
<p class="body_text"><br />
Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.<br />
</p><br />
<p class="body_text"><br />
The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.<br />
</p><br />
<p class="body_text"><br />
The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes.<br />
The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.<br />
</p><br />
<p class="body_text"><br />
Building up the competent cell stock using the home-made competent cells<br />
</p><br />
<p class="body_text"><br />
Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate number</th><br />
<th>Drug</th><br />
<th>Volume competent cells (ul)</th><br />
<th>Colony count</th><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
<tr><br />
<td>II</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>III</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>IV</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>V</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>VI</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>VII</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>VIII</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>IX</td><br />
<td>1000 X CMP</td><br />
<td>100 from T vial</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>X</td><br />
<td>N/A</td><br />
<td>100 from T vial</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.<br />
</p><br />
<p class="body_text"><br />
New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.<br />
<br />
</p><br />
<p class="body_text"><br />
Transformation number 3:<br />
</p><br />
<p class="body_text"><br />
We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.<br />
</p><br />
<p class="body_text"><br />
The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.<br />
</p><br />
<p class="body_text"><br />
After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.<br />
</p><br />
<p class="body_text"><br />
On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 11th Sepetmber</b><br />
</p><br />
<br />
<p class="body_text"><br />
Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. <br />
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.<br />
</p><br />
<br />
<p class="body_text"><br />
Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Falcon Tube no.</th><br />
<th>Content: Plate & Colony</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>2z.1</td><br />
<td>54.0</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>2z.2</td><br />
<td>28.3</td><br />
<td>2.21</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>2z.3</td><br />
<td>25.6</td><br />
<td>3.03</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>2z.4</td><br />
<td>20.3</td><br />
<td>3.16</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>2z.5</td><br />
<td>13.4</td><br />
<td>3.08</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>2z.6</td><br />
<td>15.7</td><br />
<td>2.61</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>2z.7</td><br />
<td>8.9</td><br />
<td>14.17</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>2z.8</td><br />
<td>14.8</td><br />
<td>4.08</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>5z.1</td><br />
<td>64.9</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>5z.2</td><br />
<td>36.5</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>5z.3</td><br />
<td>16.6</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>5z.4</td><br />
<td>32.1</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>5z.5</td><br />
<td>23.6</td><br />
<td>2.50</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>5z.6</td><br />
<td>53.3</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>5z.7</td><br />
<td>33.7</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>16</td><br />
<td>5z.8</td><br />
<td>50.1</td><br />
<td>2.06</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>6z.1</td><br />
<td>74.8</td><br />
<td>1.71</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>6z.2</td><br />
<td>32.6</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>19</td><br />
<td>6z.3</td><br />
<td>26.0</td><br />
<td>2.25</td><br />
</tr><br />
<tr><br />
<td>20</td><br />
<td>6z.4</td><br />
<td>42.5</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>6z.5</td><br />
<td>30.8</td><br />
<td>2.02</td><br />
</tr><br />
<tr><br />
<td>22</td><br />
<td>6z.6</td><br />
<td>27.3</td><br />
<td>2.23</td><br />
</tr><br />
<tr><br />
<td>23</td><br />
<td>6z.7</td><br />
<td>38.0</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>24</td><br />
<td>6z.8</td><br />
<td>43.5</td><br />
<td>2.10</td><br />
</tr><br />
<tr><br />
<td>25</td><br />
<td>7z.1</td><br />
<td>62.7</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>7z.2</td><br />
<td>31.7</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>27</td><br />
<td>7z.3</td><br />
<td>26.4</td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>7z.4</td><br />
<td>21.4</td><br />
<td>2.12</td><br />
</tr><br />
<tr><br />
<td>29</td><br />
<td>7z.5</td><br />
<td>13.1</td><br />
<td>2.28</td><br />
</tr><br />
<tr><br />
<td>30</td><br />
<td>7z.6</td><br />
<td>27.2</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>31</td><br />
<td>7z.7</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>32</td><br />
<td>7z.8</td><br />
<td>33.0</td><br />
<td>2.28</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the transformed cells with the ligation prepared on the 9/09<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony Count</th><br />
</tr><br />
<tr><br />
<td>Z1</td><br />
<td>12</td><br />
</tr><br />
<tr><br />
<td>Z2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>C4</td><br />
<td>1 (+small colonies)</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>17</td><br />
</tr><br />
<tr><br />
<td>pSecTag2A</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).<br />
</p><br />
<p class="body_text"><br />
Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).<br />
</p><br />
<p class="body_text"><br />
Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands:<br />
- For zeocin 2 bands: one of 1037 bp and another of 783 bp<br />
- For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Item</th><br />
<th>ul</th><br />
</tr><br />
<tr><br />
<td>DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul)</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>3</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 />
The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul).<br />
These cuts were run on a gel, next to uncuts.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 12th September<b><br />
</p><br />
<p class="body_text"><br />
Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>14</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>30</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.<br />
</p><br />
<p class="body_text"><br />
Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.<br />
</p><br />
<br />
<p class="body_text"><br />
Ligation 4<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Ligations</th><br />
<th>AA1</th><br />
<th>BB1</th><br />
<th>CC1</th><br />
</tr><br />
<tr><br />
<td>EP cut, purified pAZEC fragment (53ng/ul)</td><br />
<td>2</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>*EPD cut pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>T4 DNA ligase buffer</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>**Vol. of dilute T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>RO Water</td><br />
<td>4</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Cell Counts</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;<br />
</p><br />
<p class="body_text"><br />
** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex.<br />
These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.<br />
</p><br />
<p class="body_text"><br />
<u>Transformation 4</u><br />
</p><br />
<p class="body_text"><br />
Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.<br />
3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 13th September</b><br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin using <a href="https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction" target="_blank"> Taq PCR kit New England Biolabs</a> in 50 ul reaction volume.<br />
</p><br />
<p class="body_text"><br />
Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09<br />
</p><br />
<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>Z1.1</td><br />
<td>24.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>Z1.2</td><br />
<td>35.4</td><br />
<td>1.98</td><br />
</tr><br />
<tr><br />
<td>Z1.3</td><br />
<td>34.2</td><br />
<td>1.93</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Accomplished tasks asked by Darren:<br />
</p><br />
<p class="body_text"><br />
- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.<br />
</p><br />
<p class="body_text"><br />
- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.<br />
</p><br />
<p class="body_text"><br />
- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).<br />
</p><br />
<p class="body_text"><br />
- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.<br />
</p><br />
<p class="body_text"><br />
Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band). <br />
</p><br />
<div class="small_image_right" style="background-image:url('https://static.igem.org/mediawiki/2013/7/7b/Sep_13_UCLiGEM2013.png');height:554px;width:650px"></div><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>E&P digest (ul)</th><br />
<th>BscR1 digest (ul)</th><br />
<th>Stu1 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>EcoR1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BscR1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</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>RO H2O</td><br />
<td>1.5</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 />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 14th September</b><br />
</p><br />
<p class="body_text"><br />
Colony counts of the transformation prepared on 12th and 13th of September.<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>AA1 5xcmp</th><br />
<th>AA1 2xcmp</th><br />
<th>AA1 1xcmp</th><br />
<th>BB1 5xcmp</th><br />
<th>BB1 2xcmp</th><br />
<th>BB1 1xcmp</th><br />
<th>CC1 5xcmp</th><br />
<th>CC1 2xcmp</th><br />
<th>CC1 1xcmp</th><br />
</tr><br />
<tr><br />
<td>12th Sep</td><br />
<td>1</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>28</td><br />
<td>-</td><br />
<td>0</td><br />
<td>0</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>13th Sep</td><br />
<td>0</td><br />
<td>-</td><br />
<td>30</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>0</td><br />
<td>-</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of these minipreps – data from the next day<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>AA1 1xcmp colony number</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>26.8</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>24.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>35.1</td><br />
<td>1.90</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>28.2</td><br />
<td>1.95</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>30.5</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>28.7</td><br />
<td>1.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>35.1</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>40.3</td><br />
<td>1.88</td><br />
</tr><br />
</table><br />
<p><br />
<br />
<p class="body_text"><br />
The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.<br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.<br />
</p><br />
<p class="body_text"><br />
A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 15th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.<br />
</p><br />
<p class="body_text"><br />
From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.<br />
</p><br />
<p class="body_text"><br />
Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Volume (ul)</th><br />
<th>MMP9 4bb</th><br />
<th>MMP9 4bb</th><br />
<th>Control (no backbone)</th><br />
<th>Control (no insert)</th><br />
<th>MMP9 3</th><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>5</td><br />
<td>7.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (from K812014)</td><br />
<td>0</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (hs)</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 />
<td>10</td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Insert - MMP9</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>0</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Vial Labels</td><br />
<td>1</td><br />
<td>2</td><br />
<td>3</td><br />
<td>4</td><br />
<td>5</td><br />
</tr><br />
</table><br />
</p><br />
<br />
</div><br />
<br />
<div class="gap"><br />
</div><br />
<br />
<div class="full_row"><br />
<div class="gap"><br />
</div><br />
<p class="body_text"><br />
<b>Mammalian Labs</b><br />
</p><br />
<p class="body_text"><br />
<b>Tuesday 10th September</b><br />
</p><br />
<p class="body_text"><br />
Threw away 12 over-confluent ( aprox. 150%) plates (P15).<br />
5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.<br />
</p><br />
<p class="body_text"><br />
Added FBS and PS intp DMEM media to make new solution.<br />
</p><br />
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</html></div>Andykechenghttp://2013.igem.org/File:Sep_13_UCLiGEM2013.pngFile:Sep 13 UCLiGEM2013.png2013-10-04T23:03:04Z<p>Andykecheng: </p>
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<div></div>Andykechenghttp://2013.igem.org/Team:UCL/Labbook/Week15Team:UCL/Labbook/Week152013-10-04T22:52:57Z<p>Andykecheng: </p>
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<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 15</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 9th September<b><br />
</p><br />
<p class="body_text"><br />
Contamination control was set using 15 ml Falcons with 3 ml LB broth and 3 ul cmp or 3 ul amp.<br />
</p><br />
<p class="body_text"><br />
Retransformation of zeocin pSB1C3 ligations from 4 September and 6:1 zeocin and cmv ligations from 31 of August.<br />
</p><br />
<p class="body_text"><br />
Tubes ligations used: 5z, 6z, 7z, 8z and 9, 10 controls from 4 of September and 2z from 31 of August. These were plated and left over-night.<br />
</p><br />
<p class="body_text"><br />
LIGATION 3:<br />
</p><br />
<p class="body_text"><br />
pSB1C3 digested and purified, concentration = 25 ng/ul <br />
<br />
</p><br />
<p class="body_text"><br />
zeo digested and purified conc = 77 ng/ul<br />
</p><br />
<p class="body_text"><br />
CMV digested and purified conc = 25 ng/ul<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Zeocin (77 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [z1]</th><br />
<th>6 to 1 (mass ratio) [z2]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>6</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>2</td><br />
<td>4</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Cmv (25 ng/ul)</th><br />
<th>3 to 1 (mass ratio) [c3]</th><br />
<th>6 to 1 (mass ratio) [c4]</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>2</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>2</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Controls</th><br />
<th>5 ctrl check no circular backbone</th><br />
<th>6 ctrl check digestion process</th><br />
<th>7 ctrl (uncut backbone)</th><br />
</tr><br />
<tr><br />
<td>water (ul)</td><br />
<td>7</td><br />
<td>18</td><br />
<td>18</td><br />
</tr><br />
<tr><br />
<td>Quick ligase buffer (ul)</td><br />
<td>10</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>backbone (ul)</td><br />
<td>3</td><br />
<td>3</td><br />
<td>3 of uncut backbone</td><br />
</tr><br />
<tr><br />
<td>insert (ul)</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>ligase (ul)</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
These ligation reactions were stored at -20 degrees Celsius and used in the following day for transformation.<br />
</p><br />
<br />
<p class="body_text"><br />
Reinoculated K812014 from glycerol stocks in order to increase the stocks of backbone pSB1C3. 15 ml Falcon contained 3 ul cell culture, 3 ml LB broth and 3 ul chloramphenicol (cmp).<br />
</p><br />
<br />
<p class="body_text"><br />
These were incubated overnight and then minipreped.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Tuesday 10th September<b><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the re-transformation of ligations:<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony counts</th><br />
</tr><br />
<tr><br />
<td>2z from 31/08</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>5z from 4/09</td><br />
<td>150+</td><br />
</tr><br />
<tr><br />
<td>6z from 4/09</td><br />
<td>80+</td><br />
</tr><br />
<tr><br />
<td>7z from 4/09</td><br />
<td>100</td><br />
</tr><br />
<tr><br />
<td>8z from 4/09</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>9z - ctrl from 4/09</td><br />
<td>40</td><br />
</tr><br />
<tr><br />
<td>11z - ctrl from 4/09</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
MMP9 in cmv hygro plasmid was used to transform home-made competenet cells in order to built a stock. <br />
</p><br />
<p class="body_text"><br />
Colony count - 90% - 100+ and 10% 80 colonies.<br />
</p><br />
<br />
<p class="body_text"><br />
Biobricks (K81 and J63) as well as potential recombinant plasmid transformations (transformations of 31 August) were inoculated from glycerol stocks and minipreped in order to achieve a strong stock of pSB1C3 backbone.<br />
</p><br />
<p class="body_text"><br />
The minipreps resulted showed an average concentration of about 30 ng/ul (highest concentrations were found for one of the miniprep of K812014 biobrick - 34.6 ng/ul and for a transformation of cell vial 28: 67.6 ng/ul.<br />
</p><br />
<p class="body_text"><br />
The following day, some of these, showing high concentrations, were E and P analytical digested in 30 ul reaction volumes.<br />
The nanodrop result of the MMP9 miniprep was 42 ng/ul with 260/280 = 2.05.<br />
</p><br />
<p class="body_text"><br />
Building up the competent cell stock using the home-made competent cells<br />
</p><br />
<p class="body_text"><br />
Using the cells of a remaining cell vial of homemade competent cells, 10 LB agar plates were spread<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate number</th><br />
<th>Drug</th><br />
<th>Volume competent cells (ul)</th><br />
<th>Colony count</th><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
<tr><br />
<td>II</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>III</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>IV</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>V</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>VI</td><br />
<td>500 X CMP</td><br />
<td>20</td><br />
<td>3</td><br />
</tr><br />
<tr><br />
<td>VII</td><br />
<td>250 X CMP</td><br />
<td>20</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>VIII</td><br />
<td>N/A</td><br />
<td>20</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>IX</td><br />
<td>1000 X CMP</td><br />
<td>100 from T vial</td><br />
<td>15</td><br />
</tr><br />
<tr><br />
<td>X</td><br />
<td>N/A</td><br />
<td>100 from T vial</td><br />
<td>100+</td><br />
</tr><br />
<tr><br />
<td>I</td><br />
<td>1000 X CMP</td><br />
<td>20</td><br />
<td>60</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colonies from plate IX and X were picked and taken through the competent cell test using 250 x (4x) for drug plate.<br />
</p><br />
<p class="body_text"><br />
New inoculations (in 3 ml LB broth and 3 ul cmp) of the transformations from 4/09 and 9/09 (?) using 8 colonies from each of the following plates: 2 z, 5 z, 6 z, 7 z were prepared and left in the incu-shaker in the usual conditions over night.<br />
<br />
</p><br />
<p class="body_text"><br />
Transformation number 3:<br />
</p><br />
<p class="body_text"><br />
We transformed 7 homemade competent cells using 5 ul of each of the 7 ligation reactions set on the 9/09 (check date) as well as one more of these cell vials with pSecTag2A plasmid (concentration 30 ng/ul) as another control.<br />
</p><br />
<p class="body_text"><br />
The c3 ligation (1:3) was lost as one of the cell vials containing was taken by mistake by someone from the team.<br />
</p><br />
<p class="body_text"><br />
After the transformation procedures these cells were spread on agar plates containing 2x cmp and 2 x amp for the pSecTag2A biobrick.<br />
</p><br />
<p class="body_text"><br />
On the same day, 10 ul of z2, c4 and pSecTag2A (of potentially transformed cells) was inoculated with 2 ul cmp and 2 ml LB broth.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Wednesday 11th Sepetmber</b><br />
</p><br />
<br />
<p class="body_text"><br />
Re-inoculations of glycerol stocks of biobricks (those in pSB1C3 backbone - J63.. and K81 as well as that in pSecTag2A) and potential recombinant plasmids were made in an attempt to grow the stock of pSB1C3 backbone. <br />
There was no growth for 4 out of the 6 inoculations made for J63 biobrick and also no growth for transformed cells from original vial no. 28.<br />
</p><br />
<br />
<p class="body_text"><br />
Labeling of the 8 colony inoculations of each of the potential transformation with potentially recombinant pSB1C3 + zeocin (31 and 4/09 transformations):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Falcon Tube no.</th><br />
<th>Content: Plate & Colony</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>2z.1</td><br />
<td>54.0</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>2z.2</td><br />
<td>28.3</td><br />
<td>2.21</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>2z.3</td><br />
<td>25.6</td><br />
<td>3.03</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>2z.4</td><br />
<td>20.3</td><br />
<td>3.16</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>2z.5</td><br />
<td>13.4</td><br />
<td>3.08</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>2z.6</td><br />
<td>15.7</td><br />
<td>2.61</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>2z.7</td><br />
<td>8.9</td><br />
<td>14.17</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>2z.8</td><br />
<td>14.8</td><br />
<td>4.08</td><br />
</tr><br />
<tr><br />
<td>9</td><br />
<td>5z.1</td><br />
<td>64.9</td><br />
<td>1.70</td><br />
</tr><br />
<tr><br />
<td>10</td><br />
<td>5z.2</td><br />
<td>36.5</td><br />
<td>1.93</td><br />
</tr><br />
<tr><br />
<td>11</td><br />
<td>5z.3</td><br />
<td>16.6</td><br />
<td>2.01</td><br />
</tr><br />
<tr><br />
<td>12</td><br />
<td>5z.4</td><br />
<td>32.1</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>13</td><br />
<td>5z.5</td><br />
<td>23.6</td><br />
<td>2.50</td><br />
</tr><br />
<tr><br />
<td>14</td><br />
<td>5z.6</td><br />
<td>53.3</td><br />
<td>1.87</td><br />
</tr><br />
<tr><br />
<td>15</td><br />
<td>5z.7</td><br />
<td>33.7</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>16</td><br />
<td>5z.8</td><br />
<td>50.1</td><br />
<td>2.06</td><br />
</tr><br />
<tr><br />
<td>17</td><br />
<td>6z.1</td><br />
<td>74.8</td><br />
<td>1.71</td><br />
</tr><br />
<tr><br />
<td>18</td><br />
<td>6z.2</td><br />
<td>32.6</td><br />
<td>1.82</td><br />
</tr><br />
<tr><br />
<td>19</td><br />
<td>6z.3</td><br />
<td>26.0</td><br />
<td>2.25</td><br />
</tr><br />
<tr><br />
<td>20</td><br />
<td>6z.4</td><br />
<td>42.5</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>21</td><br />
<td>6z.5</td><br />
<td>30.8</td><br />
<td>2.02</td><br />
</tr><br />
<tr><br />
<td>22</td><br />
<td>6z.6</td><br />
<td>27.3</td><br />
<td>2.23</td><br />
</tr><br />
<tr><br />
<td>23</td><br />
<td>6z.7</td><br />
<td>38.0</td><br />
<td>2.15</td><br />
</tr><br />
<tr><br />
<td>24</td><br />
<td>6z.8</td><br />
<td>43.5</td><br />
<td>2.10</td><br />
</tr><br />
<tr><br />
<td>25</td><br />
<td>7z.1</td><br />
<td>62.7</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>26</td><br />
<td>7z.2</td><br />
<td>31.7</td><br />
<td>1.89</td><br />
</tr><br />
<tr><br />
<td>27</td><br />
<td>7z.3</td><br />
<td>26.4</td><br />
<td>1.88</td><br />
</tr><br />
<tr><br />
<td>28</td><br />
<td>7z.4</td><br />
<td>21.4</td><br />
<td>2.12</td><br />
</tr><br />
<tr><br />
<td>29</td><br />
<td>7z.5</td><br />
<td>13.1</td><br />
<td>2.28</td><br />
</tr><br />
<tr><br />
<td>30</td><br />
<td>7z.6</td><br />
<td>27.2</td><br />
<td>2.22</td><br />
</tr><br />
<tr><br />
<td>31</td><br />
<td>7z.7</td><br />
<td>-</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>32</td><br />
<td>7z.8</td><br />
<td>33.0</td><br />
<td>2.28</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Colony counts of the transformed cells with the ligation prepared on the 9/09<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>Colony Count</th><br />
</tr><br />
<tr><br />
<td>Z1</td><br />
<td>12</td><br />
</tr><br />
<tr><br />
<td>Z2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>C4</td><br />
<td>1 (+small colonies)</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>50</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>17</td><br />
</tr><br />
<tr><br />
<td>pSecTag2A</td><br />
<td>10</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Inoculations for Z1 and C4 were prepared (3 colonies for Z1 plate were picked as well as the only colony on C4 plate; all inoculatons were made in 2 LB broth and 4 ul chloramphenicol and left for 16 hours in the incu-shaker at 200 rpm, 370 C).<br />
</p><br />
<p class="body_text"><br />
Prep digest of linearised pSB1C3 from the 2013 High school distribution kit with EcoR1 and Pst1 in a 40 ul volume reaction (using 25 ul DNA and 2 ul of each E and P). This was incubated for 2 hours and then freezed. Before it was used in another ligation, it was heat inactivated at 80 degrees Celsius for 20 min).<br />
</p><br />
<p class="body_text"><br />
Analytical digest of zeocin and K812014 biobrick with Stu1 restriction enzyme. Expected bands:<br />
- For zeocin 2 bands: one of 1037 bp and another of 783 bp<br />
- For K812014, 4 bands of 1589 bp, 877 bp, 311 bp and 175 bp<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Item</th><br />
<th>ul</th><br />
</tr><br />
<tr><br />
<td>DNA zeocin (55 ng/ul)/K812014 (34.6 ng/ul)</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>3</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 />
The zeocin tube (PCR purified) of 77 ng/ul was diluted by adding 10 ul of RO water (new concentration around 55 ng/ul).<br />
These cuts were run on a gel, next to uncuts.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Thursday 12th September<b><br />
</p><br />
<p class="body_text"><br />
Prep digest of samples from minipreped inoculations 1, 14, 16, 17, 24.<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>Volumes (ul)</th><br />
</tr><br />
<tr><br />
<td>DNA</td><br />
<td>14</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>Buffer 2</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>BSA</td><br />
<td>0.5</td><br />
</tr><br />
<tr><br />
<td>dH2O</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>30</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
After EcoR1 and Pst1 digestion and running a gel of the potential backbone sources for building up the pSB1C3 stocks we decided to avoid using biobrick K812014 due to the presence of 3 bands instead of 2. Instead we decided to use J632014 biobrick as a pSB1C3 source for now on because it shows the correct number of bands and lengths of the digested DNA material.<br />
</p><br />
<p class="body_text"><br />
Prep digest of pSB1C3 (E, P digested the day before) with Dpn1 – added 2 ul of Dpn1 to the volume of 40 ul of double digested pSB1C3 and incubated it for 1 hour at 37O C. This was done in order to prepare the backbone for ligation. Also, before ligation, the tube was heat inactivated at 80O C for 22 minutes.<br />
</p><br />
<br />
<p class="body_text"><br />
Ligation 4<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Ligations</th><br />
<th>AA1</th><br />
<th>BB1</th><br />
<th>CC1</th><br />
</tr><br />
<tr><br />
<td>EP cut, purified pAZEC fragment (53ng/ul)</td><br />
<td>2</td><br />
<td>0</td><br />
<td>2</td><br />
</tr><br />
<tr><br />
<td>*EPD cut pSB1C3</td><br />
<td>2</td><br />
<td>2</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>T4 DNA ligase buffer</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>**Vol. of dilute T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>RO Water</td><br />
<td>4</td><br />
<td>6</td><br />
<td>6</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>10</td><br />
<td>10</td><br />
<td>10</td><br />
</tr><br />
<tr><br />
<td>Cell Counts</td><br />
<td>0</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
* before EPD (E –EcoR1, P- Pst1, D-Dpn1) digestion it was in a concentration of 25 ng/ul;<br />
</p><br />
<p class="body_text"><br />
** Diluted by adding 4 ul of RO water to 4 ul of DNA T4 ligase from Alex.<br />
These were left on the bench for 30 minutes and then heat inactivated for 20 minutes at 80O C.<br />
</p><br />
<p class="body_text"><br />
<u>Transformation 4</u><br />
</p><br />
<p class="body_text"><br />
Two ul of each of these ligations were used to transform One Shot Top10 Competent Cells (2004) offered by Darren. The specific <a href="http://tools.lifetechnologies.com/content/sfs/manuals/oneshottop10_man.pdf" target="_blank"> transformation protocol</a> for this type of cells was followed apart from adjustments on step 8 where after incu-shaking at 225 rpm for one hour, the tubes were pelleted for 2 minutes and the supernatant was temporarily removed from each tube and each pellet was resuspened in 100 ul of the previously removed supernatant.<br />
3 LB agar plates with 5x chloramphenicol were prepared and the 3 transformations were spread on these.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Friday 13th September</b><br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin using <a href="https://www.neb.com/protocols/1/01/01/protocol-for-a-routine-taq-pcr-reaction" target="_blank"> Taq PCR kit New England Biolabs</a> in 50 ul reaction volume.<br />
</p><br />
<p class="body_text"><br />
Nanodrop readings of the miniprep of the inoculations of Z1 from the 11/09<br />
</p><br />
<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>Z1.1</td><br />
<td>24.8</td><br />
<td>1.80</td><br />
</tr><br />
<tr><br />
<td>Z1.2</td><br />
<td>35.4</td><br />
<td>1.98</td><br />
</tr><br />
<tr><br />
<td>Z1.3</td><br />
<td>34.2</td><br />
<td>1.93</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
Accomplished tasks asked by Darren:<br />
</p><br />
<p class="body_text"><br />
- Spread straight from the glycerol stock of J632014 on 4 plates (0 x cmp, 2 x cmp, 4 x cmp and 5 x cmp) in order to build up the stocks of pSB1C3 backbone source. There was no growth the following morning.<br />
</p><br />
<p class="body_text"><br />
- Spread the remainder of transformed cells (12/09): 50 % on 5 x cmp plate while the other half on 2 x cmp. These were pelleted for 4 minutes.<br />
</p><br />
<p class="body_text"><br />
- Plan MMP9 PCR with Taq DNA Polymerase: 2 reactions making use of the 2 types of primers as well as running 2 controls (with no template).<br />
</p><br />
<p class="body_text"><br />
- New transformation using One Shot Top10 Competent Cells. For each ligation type, spread the cells 50% on 1xcmp and the other half on 5xcmp.<br />
</p><br />
<p class="body_text"><br />
Candidate recombinant plasmid (zeo + pSB1C3) analytical single digest with Stu1 (expecting 4 kb band) and BSCr1 (expecting 2 bands: one of 3435 bp and the other of 508 bp) and double digest with EcoR1 and Pst1 (expecting 2 kb band). <br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Contents</th><br />
<th>E&P digest (ul)</th><br />
<th>BscR1 digest (ul)</th><br />
<th>Stu1 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>EcoR1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Pst1</td><br />
<td>1</td><br />
<td>0</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Stu1</td><br />
<td>0</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>BscR1</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</td><br />
</tr><br />
<tr><br />
<td>Buffer 3</td><br />
<td>1</td><br />
<td>0</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Buffer 4</td><br />
<td>0</td><br />
<td>1</td><br />
<td>0</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>RO H2O</td><br />
<td>1.5</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 />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
<b>Saturday 14th September</b><br />
</p><br />
<p class="body_text"><br />
Colony counts of the transformation prepared on 12th and 13th of September.<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Plate</th><br />
<th>AA1 5xcmp</th><br />
<th>AA1 2xcmp</th><br />
<th>AA1 1xcmp</th><br />
<th>BB1 5xcmp</th><br />
<th>BB1 2xcmp</th><br />
<th>BB1 1xcmp</th><br />
<th>CC1 5xcmp</th><br />
<th>CC1 2xcmp</th><br />
<th>CC1 1xcmp</th><br />
</tr><br />
<tr><br />
<td>12th Sep</td><br />
<td>1</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>28</td><br />
<td>-</td><br />
<td>0</td><br />
<td>0</td><br />
<td>-</td><br />
</tr><br />
<tr><br />
<td>13th Sep</td><br />
<td>0</td><br />
<td>-</td><br />
<td>30</td><br />
<td>0</td><br />
<td>-</td><br />
<td>3</td><br />
<td>0</td><br />
<td>-</td><br />
<td>0</td><br />
</tr><br />
</table><br />
</p><br />
<br />
<p class="body_text"><br />
8 inoculations in 2 ml LB and 1xcmp were made using picked colonies from plate AA1, 1xcmp. 1 inoculation was prepared in 4xcmp for the singular colony on AA1 5 x cmp which grew after more than 20 hours of incubation.<br />
</p><br />
<p class="body_text"><br />
Nanodrop result of these minipreps – data from the next day<br />
</p><br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>AA1 1xcmp colony number</th><br />
<th>ng/ul</th><br />
<th>260/280</th><br />
</tr><br />
<tr><br />
<td>1</td><br />
<td>26.8</td><br />
<td>1.86</td><br />
</tr><br />
<tr><br />
<td>2</td><br />
<td>24.8</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>3</td><br />
<td>35.1</td><br />
<td>1.90</td><br />
</tr><br />
<tr><br />
<td>4</td><br />
<td>28.2</td><br />
<td>1.95</td><br />
</tr><br />
<tr><br />
<td>5</td><br />
<td>30.5</td><br />
<td>1.94</td><br />
</tr><br />
<tr><br />
<td>6</td><br />
<td>28.7</td><br />
<td>1.99</td><br />
</tr><br />
<tr><br />
<td>7</td><br />
<td>35.1</td><br />
<td>1.85</td><br />
</tr><br />
<tr><br />
<td>8</td><br />
<td>40.3</td><br />
<td>1.88</td><br />
</tr><br />
</table><br />
<p><br />
<br />
<p class="body_text"><br />
The inoculation in 4xcmp from the plate AA1 5xcmp showed a concentration of 40.1 ng/ul and 260/280 coefficient equal to 1.89.<br />
</p><br />
<p class="body_text"><br />
PCR of bb zeocin was set using Taq polymerase to make three 50 ul volume reactions and a control with no pSecTag2A template.<br />
</p><br />
<p class="body_text"><br />
A gel was run to check MMP9 PCR and zeocin PCR from 5/09 samples z1, z2, z5, z7 and control 9. The correct bands appeared apart from z2 and z7 which showed no DNA.<br />
</p><br />
<br />
<p class="body_text"><br />
<b>Sunday 15th September</b><br />
</p><br />
<br />
<p class="body_text"><br />
Prepared 9 glycerol stocks from the 9 inoculations made the day before while the rest of each of these inoculations was minipreped.<br />
</p><br />
<p class="body_text"><br />
From the plates of the day before, other 20 colonies from AA1 1xcmp plate (falcons 1.1 – 1.20) and 3 colonies from AA1 5xcmp (falcons 5.1 to 5.3) were picked and inoculated in 4xcmp LB broth.<br />
</p><br />
<p class="body_text"><br />
Ligation and transformation of One Shot Top ten Competent cells with MMP9 and EPD triple digested pSB1C3 (form high school iGEM distribution kit – label hs):<br />
</p><br />
<br />
<p class="body_text"><br />
<table><br />
<tr><br />
<th>Volume (ul)</th><br />
<th>MMP9 4bb</th><br />
<th>MMP9 4bb</th><br />
<th>Control (no backbone)</th><br />
<th>Control (no insert)</th><br />
<th>MMP9 3</th><br />
</tr><br />
<tr><br />
<td>Water</td><br />
<td>2.5</td><br />
<td>2.5</td><br />
<td>5</td><br />
<td>7.5</td><br />
<td>2.5</td><br />
</tr><br />
<tr><br />
<td>pSB1C3</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (from K812014)</td><br />
<td>0</td><br />
<td>2.5 (hs)</td><br />
<td>2.5 (hs)</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 />
<td>10</td><br />
</tr><br />
<tr><br />
<td>T4 ligase</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
<td>1</td><br />
</tr><br />
<tr><br />
<td>Insert - MMP9</td><br />
<td>5</td><br />
<td>5</td><br />
<td>5</td><br />
<td>0</td><br />
<td>5</td><br />
</tr><br />
<tr><br />
<td>Total</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
<td>21</td><br />
</tr><br />
<tr><br />
<td>Vial Labels</td><br />
<td>1</td><br />
<td>2</td><br />
<td>3</td><br />
<td>4</td><br />
<td>5</td><br />
</tr><br />
</table><br />
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<b>Mammalian Labs</b><br />
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<b>Tuesday 10th September</b><br />
</p><br />
<p class="body_text"><br />
Threw away 12 over-confluent ( aprox. 150%) plates (P15).<br />
5 P16 plates with 90% confluency were passaged and then split 1:4. Made up 5 new p17 plates.<br />
</p><br />
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Added FBS and PS intp DMEM media to make new solution.<br />
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