Team:UCL/Notebook

After the team had been assembled, several informal meetings were held. During these, introductions were made between team members, allowing everyone to get to know each other. Additionally, talks with previous iGEM team members allowed the team to gain important information and guidance on how to approach the project.

Each member of the team gave a brief presentation on an iGEM 2012 project. The projects strengths, weaknesses and approach to each section were discussed. Medical themed projects were favoured among the majority of the team.

Initial thoughts regarding project ideas were put forward. A speed discussion of ideas took place for brainstorming and basic development of ideas. The following ideas were favoured and put forward as possible project candidates:

• Weight control yoghurt

• Anti-cancer yoghurt

• Zebrafish water cleaning system for Third World

• Athletic Drug testing

• Clean Urban Air

• Neural network with glowing bacteria and fibre optics

DIY SynBio group at The Arts Catalyst were visited for feedback on the project ideas. Posters which the team had created for the group were set up within the space in order to generate feedback from members of the public during SynBio workshops. Overall the anti-cancer yoghurt idea was favoured by the majority of public and previous iGEM candidates. In general the public found the medical projects more appealing, partly because they tried to solve tangible problems that could not be mitigated soley by 'electrical' or 'mechnaical' technologies. The 'neural networks' idea gathers interest with scientists at Cancer Reserach UK and members of the public alike because applying synethtic biology to study neuroscience seems both innovative and relatively original. The zebrafish idea gathered interest due to the novel chassis. The remaining ideas did not generate as much interest as they tend to be common themes amongst iGEM team projects.

Final meetings before exams, both internally and at the Arts Catalyst. In the meantime we had taken on board our feedback, and took the best ideas from each of the most popular project to come up with a new idea that combined tackling a medical condition, with neuroscience, with using a novel chassis in an Alzheimer's disease project. The idea pool has now been narrowed down to:

• Anti-cancer yoghurt

• Zebrafish

• Alzheimer's disease

• Neural Network

Members of the group also held a probiotic yoghurt workshop for the anti-cancer project, where members of the public made yoghurt. The audience were informed about the project and opinions were gathered. Again, the fact that the porject was medical was well received, though some ethical concerns were raised so that we knew we would have to make bioethics a big part of our project from the start.

Exam period - iGEM work to commence full time after the slog through exams.

5th June

Group discussion concerning the project idea to be carried forward - favouring the 'Anti cancer project'. Roles were then assigned to team members present for intial research roles for the week:

Cancer research roles:

1. Ruxi Comisel - Proteins upregulated in cancer of the intestines. Specifically in the outer epithelial cell (enterocytes) – in microvilli. Also, what actually is... gut cancer? A general overview would be useful…

2. Khaicheng Kiew - Our chassis (bearing in mind that we will also build it in E. coli as a backup). We need to think what would make a good chassis in our case (ie. naturally found in the gut in an obvious one), and how well does the chassis fit.

3. Alex Bates - What will the killing mechanism be? A broad overview of cancer treatments is required, specifically detailing how a bacterium can administer the treatment.

Considerations:

a. The bacteria may secrete a toxin etc – how will we ensure that it doesn’t simply diffuse through the gut? b. If it is a toxin, what sort of biosynthetic pathway is required? c. Does the bacteria trigger apoptosis in the cancer cells (ie. an intracellular killing mechanism)? How can this be done from an extracellular bacterium? Perhaps beta-arrestin? d. Are there any treatments which we can take advantage of specifically because we are using bacteria? e. For example, a protein which creates holes in the cancer cells? Does using a bacterium open up the possibility of using a different cure that currently isn’t in use because we cannot target it to cancer cells – could the use of bacteria allow this?

4. Weiling Yuan - Targeting – do we use antibodies? What previous projects have used bacteria expressing antibodies? Are there any other ways of doing this? Perhaps the latching and initiation mechanisms can be incorporated into one protein?

5. StJohn Townsend - Initiation – mechanoreceptor activated upon latching? What other ways are there of doing this?

6. Tom Johnson - Past iGEM projects which we could incorporate into our own: Cancer projects, Gut projects, Protein engineering, Antibodies expressed in bacteria etc.

7th June

The team discusses findings from the initial research - further agreement that the 'Anti Cancer' project seemed to be the best idea, preparation of 'project sheets' to be sent to Dr. Darren Nesbeth for review and subsequent meetings.

11th June

looked a bit at the possible chassis species: salmonella, clostridium, helicobacter, E. coli. according to the tissue type/cancer type we shall decide which works with which. We start with E. coli in the lab.

We considered a pro-drug approach - bacterially directed enzyme pro-drug therapy which suggests that we may establish a transformed bacterial population with an enzyme capable to activate an ingested prodrug. This pro-drug would be connected to an antibody (possibly part of the tail) and would also have linking consensus sequence targeted by the enzyme produced locally by our bacteria.

From this above point Alex distinguished 2 scenarios built on the circuit sketch that he and Laia posted a while ago. These would be:

1) Kill unit produces tailed protein pro-drug (possibly tailed perforin) and signaling molecule, A. When A reaches a threshold amount, perforin and a protease to remove the confounding tail is produced, bacteria lyses and activated pro-drug acts on surrounding cells.

2) No protease is produced, because the tail can be cleaved off by matrix metalloproteases.

Goals for the end of this week:

- Alex, Andy and Weiling continue investigating possible candidates to fill in the parts for the scenarios

-Tom, KC and Ruxi make sure we have everything set up to start the work in the lab: protocol, parts etc.

12th June

Ruxi and Tom went through a general cloning protocol but then realised that the best way to prepare for the lab is to get familiarised with the iGEM distribution kits. We discovered that we are given almost everything we need in order to get it right.

Alex filled in the form with our proposal requested by Darren - we have the sequences and details of potential new biobricks.

We formulated a new proposal regarding the Alzheimer’s disease amyloid plaque degradation.

Andy searched potential cancer killer molecules:

- CD95 - Fas agonist (http://www.nature.com/cdd/journal/v14/n4/full/4402051a.html) - Tumor Necrosis Factor, Histamine - induces inflammation - HAMLET (human a-lactalbumin) - induces apoptosis - endostatin, thrombospondin - reduce cancer growth

Weiling looked at potential promotors:

- RacA (based on increased DNA damaged due to radiation) to start the killing cascade and CD95 as a potential killer molecule - Lux pR promotor - Lld promoter - Vgb promotor - HIP-1

(about gastric Oxygen levels: http://www.biomedcentral.com/1471-2180/11/96)

For promoter 1 (switches on the pro-drug and signaling molecule transcription), a very good candidate is HIP 1 promoter - hypoxia-inducible promoter which drives reporter gene expression under both acute and chronic hypoxia. It was developed in attenuated Salmonella species. Take a look here: 　 http://www.landesbioscience.com/journals/cbt/article/2951/mengesha5-9.pdf

We need to register this part!

13th June

Alex sent the 3 main project proposals to Dr. Darren Nesbeth for review.

Tom and Andy edited the wiki page adding various sections and elaborating on previously created pages.

Weiling researched on killing mechanisms being able to target hypoxic regions of solid tumors and promoters in hypoxia environments.

Catrin - General project research

Ruxi - Further researched the potential promoters esp HIP 1 and the Fas regulated programmed apoptosis.

We attended a Synthetic Biology talk by Neil Dixon, University of Manchester (Tom and Andy).

Had a general meeting for discussion of what has been accomplished so far, and the subsequent actions, which are to be undertaken by team members. Further documents were also submitted to Dr. Darren Nesbeth concerning 'team roles'. The team then began to do individual research or other activity:

Tom and Robin - Edited the iGEM wiki, added team information and removed the unnecessary tutorial information, replacing it with more useful information and streamlining the whole interface.

Weiling and Alex - Further development of circuit ideas, taking inspiration from previous iGEM ideas as well as further research into the CD95L molecule.

Ruxi and Catrin - Research into latching molecules for a bacteria to tumour interface to increase target specificity. Idea encounted from Hong Kong 2012 where Colon Cancer was targeted.

14th June

Tom - Website design for: Main Page, UCL information, Team based pages and Notebook pages

Robin - Coding in HTML for website

Ruxi, Catrin, Weiling - Further investigation of Hong Kong 2010 to see what parts may be improved or of use to the project, these were: a blue light activated promoter, how can the quorum sensing and CagA be exploited, a negative regulatory system for drug secretion.

Alex - searched for potential bacterial receptor to be modified in order to be a good target for something else in the environment/cancer cell surface.

17th June

The group had a meeting to discuss what had been achieved so far and what needed to be done today.

Tom - Continued on website design and wrote several pieces concerning UCL to be used on the website when it goes live.

Robin - Continued on website coding.

Weiling & Catrin - Researched for project sponsors and potential contacts.

Alex, Ruxi, StJohn & Andy - Continued research into the project ideas.

18th June

The group met with advisors Darren Nesbeth and Philipp Boeing to discuss the three project suggestions. The 'Neural Network' proposal was effectively ruled out due to the high risk and low probablility of project success in terms of medals.

The anti-cancer project was previously the favoured idea, but after extensive review ,the Alzheimers project gained favour due to being relatively new (and hence exciting) to iGEM compared to a cancer project, which has been done several times already at iGEM. No final decision has been made however, work has continued on researching both projects. The wiki is also still being worked on.

The team also had a social gathering: pizza for lunch.

19th June

The group continued work on all three projects in order to send improved proposals to Darren Nesbeth by the end of the day. Many professors and experts were also emailed to seek guidance, in particular for the Alzheimer's project which seems to be particularly difficult.

20th June

Tom - Prepared a presentation to be given next week about iGEM to prospective UCL students to raise interest in the engineering faculty and also the iGEM competition. After this was complete, joined the rest of the group in research. Also performed wiki coding for the team page and notebook page.

The group continued what was started yesterday: Rectifying the proposals, with both sent off at the end of the day once they were complete. A group meeting was held at the end of the day to gauge interest and vote for the most popular idea, followed by a social gathering.

21st June

Tom - Continued wiki design, coding and content uploads. Alex - Continued to redraft the proposal for Alzheimer's StJohn - Continued to redraft the proposal for Cancer

KC - Researched into other iGEM teams to colloborate with and initiated correspondence via email

The team then discusses which project was favoured. It was fairly even but Alzheimer's was slightly more popular.

24th June

Tom continued wiki design whilst the rest of the group performed research.

Once this was complete, the group had a meeting with Yanika Borg and Philipp Boeing concerning the two project ideas. Philipp favoured the Alzheimer's project whilst Yanika was somewhat undecided.

A vote was taken with Alzheimer's being the prefered project by the group as a whole once more, although consensus was not fully reached. The group agreed to decide on the project on Wednesday proceeding a meeting with Prof. Lazaros Lukas.

25th June

The group continued with general research, and also went to the Wellcome trust to seek any extra information, although this was unfruitful.

27th June

The group voted 29 -11 in favour of Alzheimer's after a meeting with Prof. Lazaro Lukas, who was helpful and seemed excited about the project. The group also met advisor Yanika Borg and she agreed with the choice. The group also scheduled lab safety training for next thursday.

28th June

Tom presented to prospective students about the iGEM project for the day.

Weiling, Alex, Andy & Catrin began to produce a 'stop motion' explanation of the Alzheimer's project.

KC, Robin and StJohn discussed lab protocols and also modelling ideas.

29th June

Tom, Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.

KC, Ruxi & StJohn – Continued work on the proposals for the meeting with Dr. Nesbeth on Thursday.

1st July

Tom – Extracted information from private wiki and shutdown performed by Philipp Boeing. Prepared for narration of stop-motion. Also discussed project proposals with StJohn and Ruxi.

Alex, Catrin, Emily, Andy – Continued work on the stop-motion project.

StJohn & Ruxi – Formed project proposals for the laboratory experiments.

2nd July

The team had a meeting with Philipp Boeing, primarily about Human Practice and which direction should be taken in terms of gaining awareness and also funding for the project. Ruxi and StJohn then continued working on experimental protocol preparation while the rest of the team visited the Science Museum to look at their Alzheimer's exhibit for inspiration on both project development and artistic direction that our human practices should take.

3rd July

The Majority of the group continued to work on the proposals as some of the components were found to be difficult to obtain or not feasible. Tom began the YSB poster design, Robin continued on the modelling proposal.

4th July

The entire group attended safety training demonstrated by Brian O’Sullivan. A meeting was also held with experts in the field concerning microglia, Jenny Reagen amongst others.

Tom continued on poster design with Catrin looking at previous posters for inspiration. Andy met with Bethan Wolfenden to talk about debating, the rest of the group.

5th July

Tom & Catrin – Worked on the poster and finished it, as well as the presentation

Alex, Andy and Weiling – Focussed on human practises, pafrticularly essay writing and documentary planning.

KC, Ruxi and StJohn – Continued work on proposals and sent completed documents to Darren.

8th July

Meeting with Darren leads to more work on proposals, particularly procurement and logistics of items required for laboratory work. The group also spent a lot of time discussing titles for the project, with ‘Plaque Buster’ and ‘Memory Guardian’ being the more popular names in an alternate voting system.

9th July

Following the meeting with Darren yesterday, the group met and rectified the experiments system to make it clearer and more achievable to obtain bronze, silver and gold medals, reducing the number of new parts required from 12 to 3 essential ones, for example.

10th July

The group sent the new proposal to Dr. Darren Nesbeth, and are to wait for a response before continuing with specific inventory/experiment write ups. Instead, the group allocated roles for this should the proposal be accepted, and then went to the gallery of surgery to investigate cranial injections, and the implications and feasibility of this form of surgery.

11th July

The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.

12th July

The group spent the majority of the day preparing for the Young Synthetic Biologists event, with Tom, Alex and KC practised their presentations, with the whole group contributing to the poster and also deciding on the working title, although this was unsuccessful.

13th July

YSB Day 2: Collaboration continued between teams for feedback and suggestion purposes. Tom and Alex initiated the creation of a national SynbioSoc so it easier for iGEM teams to communicate ideas and generally collaborate for both this year and the future. Tom also announced the iGEM football tournament, which was met with enthusiasm by other teams.

15th July

First day of lab, under instruction by Dr. Darren Nesbeth and Yanika Borg, the team were shown various items in the labs and how to use them, with emphasis on good laboratory practice at all times. The team also met up with Oran and FongYi to discuss how the artistic side of the project will be undertaken. Oran and FongYi joined the team.

16th July

Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team created ‘minimal agar’ plates to grow W3110 E. coli cells on. The cells were left to incubate overnight for a 16 hour period.

KC, Alex & StJohn – Worked on primer design for the PCR reactions planned. Difficulties with finding flanking DNA sequences were encountered.

17th July

Tom, Catrin, Andy & Weiling - Under supervision from Yanika Borg, the team looked at the cell cultures in the morning and discovered that the cells had not grown, so came back in the afternoon and noticed growth on 2 of the 5 plates. Further incubation of 17 hours was agreed upon.

KC & Alex – Started mammalian cell lab induction.

The team then met with artists to further develop the branding of the whole project.

18th July

Tom, Catrin, Andy & Weiling – Lab experiment with Yanika Borg – Selection of colonies then resuspension into growth media, followed by incubation until 10:00 tomorrow.

StJohn & KC – Primer design for the PCR protocols

Alex – Continued work on ethics and feasibility report & bioinformatics

19th July

Tom, Catrin, Andy & Weiling – Continued Lab experiments with Yanika Borg – Re-suspension & centrifugation of colonies.

StJohn & KC – Primer design for the PCR protocols

Alex – Continued work on ethics and feasibility report & bioinformatics

22nd July

Meeting with Darren reveals that primer design needs to be reconfigured, and that the strategy for Gold is currently not acceptable, so this will be worked on. We won the inter-UCL award for best wiki of July. StJohn worked on primers and KC worked on protocols.

Tom, Catrin, Andy & Emily performed bacterial labs, using transformation skills.

23rd July

Tom, Catrin, Andy & Emily performed bacterial labs once more, repeating yesterday’s experiments due to a failed transformation.

StJohn did more rectification work on primer design. KC searched for any possible molecules which could be used as an alternative molecules that naturally exist in the brain as replacements for auxin detection system.

Weiling & Alex went to KCL (Institute of Psychiatry) to interview professor John Powell, an expert in the field of Alzheimer’s diseases, and other brain related diseases.

24th July

Until the 26th of July the bacterial lab work did not get any further. Several transformations were performed but neither was successful. After these trials, the decision of making new competent cells was taken.

The entire team was sent the information regarding mammalian lab aseptic techniques. StJohn analised an article on Microglia function in Alzheimer’s disease.

Alex gathered more information regarding main transcription factors/promotors we could use for detecting the oxidative stress caused near plaques.

The team decided to meet over for a barbeque on the 7th of August.

25th July

Oran came to the lab and was introduced to the lab routine and to the activities on going. The team met again in the Student Anatomy hub to continue research on useful articles.

26th July

A summary of the week lab work:

- We have made stocks of all constituents needed to grow cells (E. coli W3110) and have a stock in the -80C cold storage.

- We attempted transformation (p1313) on three separate occasions but it failed each time (although controls worked as expected).

- We used Yanika's personal cell stock of W3110 and performed the transformation successfully.

- Therefore today we remade the constituents needed at the start, we will perform plate streaking etc. after the weekend, and hopefully have more success with transformation as well.

The following biobricks were ordered BBa_1712004, BBa_K812014, BBa_J63008. They’re supposed to arrive through UPS service by the 31st of July.

29th July

An important day for our team! The project name “Spotless mind” was chosen!

The MathWorks license for the 2013 iGEM student competition has been created.

The Biobricks from the iGEM HQ arrived today, which includes a mammalian plasmid backbone and 2 auxin signalling parts.

30th July

The entire team is involved in organising the speed debate taking place tomorrow, 31st. FYi and Oran produced a nice debate poster and a new logo!

31st July

We organised a neuroethics themed Speed Debate at Print Room Cafe, UCL. We started preparation such as buying refreshments, setting up the venue, printing survey sheets and poster at 4pm. At 7pm, guests started to arrive. Over 90 participants attended the speed debate. Dr. Howard Boland, Alex Bates, Philipp Boeing and Shirley Nurock from the Alzheimer's Society spoke at the speed debate.

The event was a success, many guests stayed to discuss further and alot of interests were received regarding the progress of our project. We cleaned the venue and wrapped up at 10.30pm

1st August

Bacterial lab had good results today in the preparation of a new stock of competent cells. In the evening we celebrated the success of the speed debate.

2nd August

Stjohn designed the linkers for the Mammalian Oxidative Stress Inducible Promoter. The team met to discuss fundraising ideas somehow making use of [kickstarter.com]. A starting idea: brain-with-plaques-for-sale.

We came up with the idea of a Memory Lane, where people could upload a photo of one of their memories and write a small description about it.

Alex suggested a collaboration with Westminster iGEM team regarding the speed debate idea.

5th August

Snapshots of the team members were taken!

The team worked on the abstract which must be uploaded shortly on wiki as the deadline is on the 9th.

Alex contacted the Imperial iGEM team regarding an eventual collaboration.

6th August

Rob invited the team at 12 noon in the Anatomy Hub to discuss about the wiki design in order to make sure that all the ideas about this matter are taken into account.

7th August

Barbeque evening, venue Wilkins Roof Garden!

Prof. Eli Keshavarz-Moore was our guest and at 3 pm we also had the chance to present our project. (venue: Malet Place Engineering LT 1.03)

8th August

The team discussed about the work on zeocin,pA-f1-Zec biobrick, which will indeed be an improvement of BBa_J176124 because:

i) it gives most of the functionality of BBa_J176124 but is compatible with standard assembly

ii) it allows people to simply insert a PROMOTER-ORF fragment upstream of a pA to give an expression cassette for the ORF of interest, and a ZEC to select stable transfectants.

9th August

Project description is up on Wiki!

Darren gave us a visit at the lab to check if everything is O.K. with our work and enthusiasm. The requested batch of biobricks arrived as glycerol stocks.

The team discussed about Kickstarter crowdfunding and planned to launch the Memory Lane/Map thing WITHOUT getting people to pay. We will get people to upload their best memories in different forms and potentially do some beautiful art with it like the Memory Palace FYi suggested.

12th August

We had a strategy chat at the lab with Darren.

FYi drawn the wiki background for the diary section. She also made the illustrations for the T-shirts.

The team also debated on the wiki design and a consensus was reached regarding the site map, default banner, logo.

In 'Memory Lane', we are going to ask people to 'leave one strong memory' on one page whether in text or pictures. These will be done anonymously but they will leave their emails with us so they will be notified when the 'compilation' is up.

The website came to life today!

13th August

Alex and Oran came up with the idea of a Creative writing competition.

FYi, Robin, Alex and Stjohn and Oran focused on wiki building for the weeks to come while the rest of the team worked in the Bacterial Labs.

14th August

The advertisement for the competition was written and the competition was launched. More details about the outcome can be found on the ‘Competition’ subsection.

Met the Westminister team to discuss about the potential modelling collaboration. It was a nice gathering.

15th August

Continued intensively planning and brainstorming for the design of our wiki, especially on the front page design.

16th August

17th August

18th August

19th August

Alex advertised the writing competition on prizemagic.co.uk.

Stjohn released a new set of rules for managing wiki content in order to make work easier before the wiki freeze.

20th August

The actual work on the main poster on the frontal page started. FYi produced the first sketch and the team gave feedback.

The members’ Profiles are ready to be uploaded on wiki!

21th August

The lab was closed in the morning, however in the afternoon the Bacteria Team prepared selective plates and selective media in order to culture the last arrived biobricks from the HQ. Darren assisted us.

The linkers designed by Stjohn: IGM Ox L1, L2, L3, L4 as well primers for cmv promoter were ordered.

22th August

The first Creative Competition Entry! Yey! Thank you!

The atmosphere in the Bacterial Lab became slightly more cheerful. The amplification of zeocin from the 2 types of ordered primers was successful as well as the digestion of K812014 and pSB1C3 and pSB1A3. We decided to use the zec bb F,R primers for the further amplification of zeocin.

The Zeo kill curve was derived, a concentration of 150 ug/ml was used.

23th August

The main poster for the front page was finalised. Well done FYi!

New submissions for the Creative writing! Lonza confirmed a sponsorship of £1, 207. Happy Happy Joy Joy! Well done Weiling!

26th August

The lab was closed today hence we all focused on the wiki content.

The front page poster background - wasteland was completed.

27th August

28th August

The Biosafety forms were filled in as necessary. These must be signed by Darren before the 30th.

We met Darren at 4 pm in the lab to discuss about the biobrick processing.

29th August

We considered the strategy to deal with the linker region. First step is to achieve the annealing of the oligonucleotides making up this linker. We're still waiting for these sequences.

Agreed on the final design of the T-shirts. We're aiming to order them as soon as possible.

30th August

We uploaded the first samples of memories on the Memory Lane page.

1st September

The Bacteria Team is living some intense moments! The first transformation of the zeocin ligation took place yesterday and we're all very optimistic! We're about to know the results of this zeocin cloning on the 2nd, the latest the 3rd.

2nd September

We finally received the oligonucleotides needed for the linker region! We can now start the cloning plan for this biobrick.

3rd September

We started to think which type of poster would be the best idea for the Jamboree presentation.

We met Darren at 4 pm to discuss about the cloning strategy for MMP9.

4th September

We used SurveryMonkey in order to make a decision on who should present at the Jamboree.

We reached a consensus for Alex, Tom and Casey to carry out this precious job for the team.

5th September

We decided that the best option as the background colour for the T-shirts would be white.

6th September

HQ replied about zeocin resistance biobrick. It will count as a new part. They also confirmed our attendance to the Regional Jamboree. Lyon, here we come!

Alex produced a first draft of the poster while the other gave him feedback and FYi offered to take care of the actual design.

9th September

Today Darren visited us at the lab and brought us MMP9 which was used to transform our competent cells.

A new ligation for zeocin was prepared and competent cells were transformed with it.

10th September

All the photos of the team members and supervisors were mounted on wiki.

We had another discussion with Darren who advised us to test again the chloramphenicol and also to prepare more competent cells. He also reminded us to always use pSecTag2A as a control when minipreping.

11th September

12th September

We agreed on the final details of the

13th September

14th September

15th September

16th September

17th September

18th September

19th September

20th September

21st September

22nd September

23rd September

24thSeptember

25th September

26th September

27th September

28th September

29th September

30th September