Team:UCL/Notebook/June

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

- 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

gastric Oxygen levels

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.

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.