Team:Imperial College London/Diary

Our first day of iGEM was also our first day of getting used to being filmed by Kelly Neaves and Dominic Rees-Roberts, who, having already completed PhDs in the biological sciences, were on a course in Science Media Production at Imperial. Like us, they were funded by the Wellcome Trust.

Two students from the RCA called David Benqué and Gerrit Kaiser ran a Creativity Workshop on Monday afternoon which really helped us, as our brainstorming sessions were becoming less and less productive as the day wore on. They demonstrated how important it was to keep a sense of perspective when coming up with ideas.

On Monday afternoon, we were given some mini-lectures by directors of the different groups within the CSynBI. This helped us to know who to get in contact with when we needed help on certain areas of our project.

We participated in a Human Practices Workshop on Tuesday, which was organised by Susanna Finlay, a PhD student at LSE who also works in the CSynBI. We first discussed some of issues associated with synthetic biology, and were then divided up into small groups and given a case study of a synthetic biology application. After discussing some of the ethical, legal and social implications (ELSIs) of each application, we presented our ideas to the other groups. This workshop started us thinking of our design in terms of biosafety and what kind of application we wanted to develop.

On Tuesday afternoon we each did a mini-presentation of our favourite past iGEM project, which enabled us to get a feel for the iGEM competition, while at the same time allowing us to see what is feasible in terms of getting results in a short space of time.

Our first meeting with our advisors was on Wednesday morning, and really pushed us to make decisions when it came to which ideas we wanted to develop further, and which ones we had to discard. The feedback from the advisors was generally positive, but at the same time it forced us to rule certain ideas out.

Thursday was spent on further development and research of our ideas.

On Friday, we had another meeting with the advisors. We discussed our progress so far, and also started talking about other aspects of our project, like Human Practices and modelling. This was our first Cake Friday, and Kyasha managed to set the bar sky-high with a delicious mint chocolate cake.

The presentations we had been developing in our pairs during Week 1 were given on Monday afternoon to the rest of the team. The pairs and topics were as follows;

• Florian & Piotr - Standardisation
• Maddie & Ben - BioBrick Assembly Techniques
• Harriet & Anita - Characterisation Methods
• Nicolas & Kyasha - Networks in biological systems
• Kirill & Wolf – Human Practices

Our first Journal Club, held by our supervisor Chris Hirst, took place on Tuesday. This was followed by a Modelling Workshop with Dr Guy-Bart Stan, a lecturer in Synthetic Biology at Imperial. We also completed an online Wiki tutorial so that we could use the Wiki more effectively and keep all the advisors up-to-date with what we had been doing. The second session with the RCA was on Tuesday afternoon, and this time it focused more on the media attention around synthetic biology and public engagement. In small groups we looked at tabloid newspapers, and then tried to make our own headlines for a synthetic biology news story.

On Wednesday, we had a meeting with the advisors in order to write a project description, which was due by Thursday. We also took the opportunity to get some useful feedback about the project and what direction we should be going in.

On Thursday, we were lucky enough to have a mini-lecture given by Dr Martha Betson, who works on schistosomiasis at the Natural History Museum. Her extensive knowledge of working in sub-Saharan Africa enabled us to have a realistic view of how useful it would be to detect schistosoma in water.

The Human Practices Panel Discussion, with Claire Marris, a Senior Research Fellow at LSE, took place on Friday. Also present were members of the RCA and CSynBI. This session allowed us to analyse the many social aspects of our project, which led us to alter its design in certain ways. For more details, see the Human Practices Report.

For our second Cake Friday, Ben brought a cheesecake and a chocolate roulade in. A very wise choice indeed.

We met with Murray Selkirk, Professor of Biochemical Parasitology at Imperial, on Monday morning. He gave us lots of advice on the elastase that is produced by schistosoma cercariae. We also discussed the sensitivity of the detection system, and the ways in which we could test it. We stayed in contact with Professor Selkirk for the rest of the summer, and he gave us some really helpful guidance with the project, for which we are very grateful.

On Tuesday, we started to drill down on certain aspects of our design which needed further research before we could proceed. Dr Tom Ellis took the Journal Club on Tuesday afternoon.

Thursday was a busy day, involving meetings with Dr Leak (reader in Applied Microbiology) and Professor Filloux (from the Centre for Molecular Microbiology and Infection) who gave us some really useful guidance on various parts of our project, specifically the detection and signaling modules.

We spent the rest of the week researching exactly how our system could work, and at what points it might not work. This was pretty tough, as we had to think realistically and often do away with components that some members of the team had been researching for days.

Piotr baked an apple sponge on Friday!

On Tuesday morning, our very own supervisor, Chris Hirst, gave us a tutorial on how to use PlasmaDNA for In silico cloning. We then talked about the approximate time frames for standard assembly.

Tuesday night was our first movie night! We were all in need of a break from work, so we ordered pizza, watched a film and chilled out. For the rest of the week, we set about putting together our DNA cassettes for synthesis, and we also defined the genes in the B. subtilis genome into which we would integrate the DNA cassettes. We also researched Dif sites, which would allow us to remove antibiotic resistance genes after the cassettes had integrated into the genome (see the Human Practices Report for more information on this). We also started modelling how fast our system would actually be, depending on whether it was a one-step or a two-step amplification. Initially, online sources were explored for guidance on how to start modelling. Introductory lecture given by Dr Guy-Bart Stan proved being very useful. From there, first steps towards creating a model using Michaelis-Menten kinetics were made.

We presented our progress so far at a meeting on Thursday. We explained why we wanted to use the Dif sites in our DNA cassettes, and what DNA we wanted to get synthesised.

On Friday, we started researching catechol 2,3 dioxygenase (C2,3O), which is an enzyme that produces a yellow product very quickly on addition of its substrate, catechol. It is coded for by the gene XylE and was already in the registry, so we started thinking about it as a potential candidate for our output.

Anita made a fantastic banoffee pie for Cake Friday and Wolf held a much needed party on the Saturday. He cooked amazing burgers and we had our first game of horse-racing!

We then started looking at ways of making a fusion protein with C2,3O and a tag, such as glutathione-S-transferase (GST). We had a meeting with Dr James MacDonald, who gave us some useful advice about which terminus of C2,3O to fuse the tag to, and what we should be thinking about in terms of a linker.

On Wednesday morning we had a meeting with our advisors to discuss the assembly strategy and the ways in which we would test each part of our system. We also went over what modelling we had done, and how this was influencing our design.

We visited the RCA on Wednesday afternoon, and were able to see the workshops and a rapid prototyping machine. We then had a really useful discussion with David and Gerrit on Human Practices. It was great to see our project from the perspective of a designer, and this made us think about the social aspects of a parasite detection kit.

There was a meeting with Dr Matthieu Bultelle on Friday to talk about the problems we had been coming across when trying to model the system. The main issue was that our system was recognised not to obey the Michaelis-Menten kinetics. It is because Vmax is proportional to overall enzyme of concentration that is varying in our system. Furthermore, in our system the substrate is not in a great excess over the enzyme. Hence, it was decided to try develop model from the first principle (representing all chemical reactions by equations) using only Law of Mass Action. Problem of finding constants needed for equations was discussed as well, as this has shown to be task of great difficulty.

Harriet attempted millionaire shortbread for Cake Friday, but it went a bit hard… So instead we had profiteroles! Yum. We finished the day with a drink in Eastside with some of the advisors.

The Modellers have been working throughout the week on implementing the output amplification model. They have been exploring different environments to do it in, such as: MatLab or TinkerCell. They decided to go with MatLab to have better understanding of the observed behaviour. They have decided to model 1,2,3 step amplification systems. They again run into a problem of determining rate constants required for the models.

On Thursday, we had a meeting with Professor Paul Freemont, co-director of the CSynBI. He made us realise that we could have been working much more efficiently, and advised us to set objectives for each week. His words were taken as constructive criticism and we actually ended up working much more successfully after the meeting, by dividing up topics to research and communicating with each other more effectively.

After the meeting, we split up into dry lab and wet lab teams. Within the dry lab team were the modellers, Anita and Piotr, and Ben, who worked on the Wiki and other design strategies for the project. Within the wet lab team, there were 3 different pairs, which each started working on a different part of the assembly strategy. Nick and Maddie formed the XylE Team, Kirill and Kyasha formed the Vector Team and Harriet and Florian made up the Surface Protein Team. Wolf and Ben were also given the task of overseeing the project so that everything ran smoothly.

The XylE team started work in the lab on Thursday afternoon, transforming E. coli with a vector containing XylE, and working on the promoter, part J23101.

The Vector Team had the task of assembling the AmyE vector and the PyrD vector, which could then be used to integrate any piece of DNA directly into the B. subtilis genome, and at the same time, disrupt essential genes (see the Human Practices Report for more information). We used these vectors for testing constructs as well as the final assembly. Kirill decided to take on the AmyE vector, and Kyasha assembled the PyrD vector.

Nick brought in Krispy Kreme donuts for Cake Friday!

The XylE team miniprepped plasmids containing the XylE gene, which was used for assays for C2,3O activity. They also used 3A assembly to join the J23101 (promoter), XylE and pSB1C3 together. At the same time, they started the construction of the GFP-XylE fusion protein.

The surface protein team spent the week cloning the double terminator (B0014) part into pSB1C3, which will be needed at various points in the assembly strategy.

On Tuesday we were lucky enough to meet Jay Keasling, who was really inspiring and very positive about our project. We were plied with drinks by our advisors (which we had no objection to) and then ended the night with a few drinks at the union.

The modellers had a busy day on Tuesday, with meetings with Dr Bultelle and Dr Stan to discuss the abnormal behaviour of the output amplification model as well as the ComD receptor signalling. Once viable reaction rate constants have been entered into the output amplification model, it started exhibiting strange behaviour. Sometimes it was showing negative concentration values! When discussing the issue with advisers, several solutions were suggested. Finally, it was found that our system was stiff and required different solver. Initial steps were made in direction of developing the display protein signalling model. This was withheld towards the end of the week, since it was pointed out that sensitivity analysis should be performed on output amplification model to determine the key constants that we should aim to get measured in the lab.

Kirill bought a strawberry cheesecake for Cake Friday!

The Surface Protein Team did a PCR to obtain the LytC cell wall binding domain (CWBD) from the B. subtilis genome for ligation into pSB1C3 and subsequently transformation into E. coli. They also started cloning pVEG.

The modellers had a meeting with Dr Bultelle regarding further improvement and development of the output amplification model and getting opinion on the developed protein display model.

On Thursday night, we all went to Nick’s house for pizza and a few drinks. Kirill graced Earl’s Court with a midnight run and Piotr showed off his amazing dancing skills.

For Cake Friday, Florian made a very impressive homemade cheesecake. Clearly his cooking skills are as good as his lab skills!

On Wednesday, we had a meeting with our advisors to discuss the characterisation of certain parts of the system.

The XylE Team ligated the promoter-XylE insert into the pSB1C3-B0014 vector (which was previously prepared by the Surface Protein Team). They also worked on the catechol assays, determining the optimum absorbance wavelength for the coloured product, the optimum cell density and the most appropriate catechol concentration for doing the assays.

The Cell Surface Protein Team had some problems, such as a failing to transform E. coli with pVEG. They did manage to get transformants for the LytC CWBD, but after checking if it had inserted in the correct orientation, they found that it had inserted in the wrong orientation in each colony that they tested. Working with pVEG was also tricky, because it kept disappearing after gel purification because it may have been below the threshold.

On Thursday, we had a visit from Helen Craig, who works for Furnace TV (a television production company), who seemed really interested in our project. We took her on a tour of the lab and showed her a catechol assay.

The Surface Protein Team continued to work with pVEG and the LytC CWBD.

On Friday we had a meeting with our advisors to show them the results from the catechol assays.

Wolf bought Krispy Kremes for Cake Friday! Then we all went to Eastside with some of the advisors for the last bit of filming that we’d have to do!

The Surface Protein Team finally had some good news this week; pVEG and the LytC CWBD were successfully ligated and transformed into E. coli.

On Wedneday night, Kyasha threw an awesome party. Both Wolf and Piotr sang for us and some of the guys ended up showing off how many push ups they could do. Unfortunately, the girls didn't seem that impressed.

She also made a construct with XylE terminator in pSB1C3 ready to accept any promoter because we were waiting on Com and pVeg at the time. Florian digested the surface protein linker genes to confirm their presence in pSB1C3. He then midiprepped the successful transformants. Piotr PCR'd out XylE in the reverse orientation to insert into the LacI construct.

Wolf did another growth assay to see the effect of catechol on the growth E. coli cells, and got fantastic results! Kirill did loads of minipreps of things from synthesis.

On Wednesday, we held our first Synthetic Biology School Workshop at St Augustine's High School in Kilburn, which was really good fun and informative for both the school students and us. The second and third workshops were held at Harris City Academy in Crystal Palace on Thursday. See the [http://openwetware.org/wiki/IGEM:Imperial/2010/Ethics Human Practices] page to find out more!