On our first day back the team and Dr. Anne Smith had a meeting where we discussed our project idea of creating a kill switch by using antimicrobial peptides. We proceeded to divide the different tasks amongst ourselves and came up with an action plan for the next few weeks. The purpose of this was twofold, to make our team more efficient and have some sort of way of measuring progress.

We made sure that we all knew the iGEM 2011 Judging Criteria and recorded all deadlines posted on the iGEM website on our team board.

The lab team familiarized themselves with the lab where we would be carrying out our experiments over the summer and we did some simple lab chores such as autoclaving various jars and bottles, preparing Luria broth for plating and storage and learning about different lab procedures necessary for our experiments.

Max began work on the St Andrews team wiki, while Christina began creating a list of an assortment of labs and companies that we could contact for sponsorship.

Charlie began work on the Project Description while Sam and Max continued work on the team wiki. Lamya and Christina collected the notes of previous IGEM meetings that took place during the semester and built mind maps to be put on the wiki. The lab team prepared overnights of E. coli in LB so that they could run a practice transformation and gel electrophoresis later in the week.

We completed the Project Summary form and Safety Proposal and put them on the team wiki. The team then concentrated on finishing with the pre-summer meeting material. After completing the sponsorship proposal, Christina began emailing potential sponsors.

In the meantime the lab team carried out practice transformations on the E. coli from the overnights. They used a biobrick which provides resistance to ampicillin in order to learn correct antibiotic usage.

Charlie and Ogaga began researching various constitutive and positive promoters, in order to complete our basic biobrick sequence. Because our decided coding gene region was so small, it was decided that it would be feasible to order the promoter, ribosome-binding site, coding region, and terminator from a DNA synthesis company.

We booked the flights and taxis for the trip to Norwich and created a St Andrews iGEM Gmail, Facebook, Flickr and Twitter accounts. The latter would be used to communicate with other iGEM teams throughout the project.

The lab team made more overnights of the transformed E. coli colonies for the experiments to be carried out on the following day. Chris and Lamya continued to contact the 75+ potential sponsors to acquire us reagents and money for later use in the lab.

The lab team ran a mini prep, a restriction digest and a gel electrophoresis. The results of the gel electrophoresis showed that no DNA was present in our gel so we discussed the possibility that we had let the DNA sit in the wells of the gel for too long, causing it to sink through the gel. In order to corroborate this we decided to run another gel on Monday morning.

The team continued contacting sponsors, receiving some positive responses. For our human practices project the team brainstormed various ideas such as conducting surveys across various topics, public engagement, etc.

The lab team re-did the gel electrophoresis which had previously shown no DNA present, with a smaller time gap between preparing the gel and running the DNA on it. Our experiment was successful and the results confirmed our hypothesis. Charlie and Ogaga further explored different promoters and regulators and completed the basic biobrick sequence for the project. With this done, Dr. Smith was able to order the necessary sequence from the California-based DNA synthesis company, DNA 2.0.

Christina and Lamya continued emailing and calling sponsors while Max continued structuring the team wiki.

The team discussed and researched conjugation and drug-delivery as potential applications of our project. We then explored the IGEM registry for biobricks that could help us test the feasibility of these applications.

Later on we brainstormed ideas for our human practices project and discussed some potential questions that could be addressed.

As a part of our human practices project, the team decided to explore the possibility of conducting a meta analysis on the data provided by the human practices projects of previous iGEM teams, in order to find some correlations between the projects of different universities around the world.

Sam, Ogaga and Lamya collated the data for all the 2010 human practices projects and ran some basic statistical analysis tests. They concluded that the projects were too divergent to show any significant trends that could be used to draw conclusions, leading us to abandon the idea of using this idea as our human practices project.

We prepared and practiced the presentation we planned to give at the UK iGEM Meet-up in Norwich on Monday. Max continued working on the wiki. Charlie and Ogaga concentrated on designing some experiments protocols to test the final project once our kill switch was created.

The team discussed further potential ideas for the human practices component of our project, and we agreed on an innovative project to hold a debate at the World Schools Debating Championships, being held 20 minutes away in Dundee in 3 weeks time. This project however required support external event organizers, so we drew a clear human practices plan and contacted the relevant parties.

Day 1 and 2:

The St Andrews iGEM team flew to Norwich to participate in the UK iGEM meeting, where we presented our project idea and progress to date to 7 other university iGEM teams (including Cambridge, University College London, Edinburgh University, and more). At Norwich, we were able to interact with other iGEM team members and were given advice by various professors that had volunteered their time to answer our questions. Overall, it was a rewarding experience!

Day 3:

We spent the morning discussing our time in Norwich, the lessons learnt and their possible applications in relation to our project. In Norwich, there was a specific speech on Human Practices that caused us to pause and reevaluate our original debate idea. We agreed that supplementing this project with reflective essays would benefit our chances of receiving a gold medal, while staying true to the real meaning of what a Human Practices project should be: introspective.

Day 4:

The team had a morning meeting with the advisors being present. Charlie wrote up a summary of the team’s achievement in the last week. Sam re-contacted the head of the World Schools Debate Championship to confirm our place in the program.

Day 5:

The team made plans for the debate and focused on planning the experiments for our Drug Delivery Proof and checking the viability of conjugation based on our available time and team size. A lot of research was also done into conjugation functionality in vitro.

Day 1:

The team had a morning meeting with the advisors present. Meanwhile, Charlie met with Dr. Peter Coote, a resident molecular biologist who had written several papers on antimicrobial peptides, for advice on the project and the feasibility of intracellular antimicrobial peptide production as a viable kill switch.

We brainstormed some more on our human practices project focusing the the idea of a review of past iGEM teams, rather than simply their HP projects. We came up with a number of variables, including projected budget and budget at time of registration, number of biobricks submitted, number of advisors and advisor/student ratio, and many more, in order to eventually find correlations between the data that would reveal what it takes to get gold in iGEM, or even whether iGEM is truly a fair competition. and began extracting data from the wikis of previous iGEM teams.

Day 2:

The team focused mainly on the working to gather data for the iGEM review. We all spent time researching online to find various figures.

Day 3:

Ogaga, Lamya and Sam continued updating the spreadsheet. The team also agreed to collaborate with the University of Dundee’s iGEM team concerning the debate and made plans to set up a meeting to discuss the details of the debate in person. Chris and Max continued reviewing papers to find figures for their modelling.

Day 4:

Ogaga, Lamya and Sam continued updating the spreadsheet. The team had our bi-weekly meeting with our supervisors. Chris and Max reviewed modelling challenges, while Sam, Max and Charlie planned a trip to Dundee for the debate collaboration with The University of Dundee iGEM team.

Day 5:

We got DNA that we had ordered from iGEM, as well as our sequenced Biobrick DNA from DNA 2.0. Charlie tested a potential lysing agent (SDS) for the Kill Switch Proof protocol, but found that the bubbles it created were interfering with the experimental absorbance readings. The modelling team continued to read papers while Ogaga, Lamya and Sam continued gathering information for our human practices component.

Day 1:

We had our bi-weekly meeting. The modelling team read papers and found a few regarding the σ 70 ribosome, which relates to transcription rates. They emailed John Mitchell about pBAD promoter advice and transcription details. The human practices team continued gathering information for the iGEM review. We also extracted DNA from the DNA 2.0 shipment.

Day 2:

We transformed our Biobrick sequence into supercompetent E. coli, as well as plated normal supercompetent E. coli for use as control experiments in the near future. Max and Chris tried to create a hybrid system of equations, taking components from last year's modelling project, as well as components other past iGEM teams such as Berkeley 2008. At the end of the day, they were still left with a lot of unknown constants that they had been trying to source from papers.

Day 3:

Sam, Max and Charlie visited Dundee to make plans concerning the debate collaboration. They met with John Urch, head of the Revealing Research Dundee, and members of the University of Dundee iGEM team, to discuss the format of the debate and the details of the proceedings. Ogaga made overnights to test our promoter functionality the next day. Lamya and Ogaga continued gathering information for iGEM review.

Day 4:

We had our bi-weekly meeting with our advisors. Max and Chris received modelling help from our advisors, and plowed ahead into more papers to try and find the constants required for their models. Sam and Charlie did a test run of the Kill Switch Proof, which proved to be fruitless.

Day 5:

We got further responses from our sponsors. BioSilta gave us a voucher worth £500 so we could order anything below that value from their catalogue. The modelling team met with Chris Hooley and decided that their system . They also concluded that adding another set of equations relating the pBAD repressor (AraC) and pBAD promoter concentrations will be needed. They still have to find the production/degradation/dissociation rates for the relevant reactions and may brute force the data to tell us which values, in specific ranges, will optimize for protegrin-1 production.

Day 1:

We had our bi-weekly meeting with our supervisors. We responded to Biosilta with a list of the things we may need in the lab, including more supercompetent E. coli and various reaction reagents. The modelling team looked at compiling the last set of equations on pBAD inducer and pBAD promoter but found extremely limited information in this area. There were discussions and the team decided to e-mail Dr Roger Griffiths, an expert on enzyme kinetics, a contact obtained from Sarah. The database was also completed today, a huge success for the human practices team. Chris sent out emails to some statisticians to help in the analysis of our data.

Day 2:

The modelling team found an error in the original set of equations. They discovered that the repressor wasn't separated from the promoter, although it’s concentration does change. Also, the Arabinose+AraC complex concentration linked to the promoter ‘strength’, and by extension, the transcription rate. Ogaga and Sam performed a miniprep and digestion of our Biobrick extracted from E. coli, while Charlie looked at primer construction for use in creating our Drug Delivery Proof. The team also worked on the WSDC presentation and started collating quotes and ideas for the cons of synthetic biology (as Dundee was assigned the pros of synthetic biology).

Day 3:

The modelling team had a meeting with Chris and John to discuss the current state of modelling and realised that they have a few constants but need to work on the arabinose system/equilibrium inside the cell. They also decided to use the MATLAB platform to perform simulations. In all, they have a general setup with a variety of ‘ranges’ for constants. Sam and Charlie continued to create the presentation for Sunday. Sam also found a working digestion protocol.

Day 4:

The modelling team looked through KEGG to see the pathways that are affected by arabinose in the cell. Max continued looking at MATLAB to see how to set up equations. We also sent off our logo to Genescript for our t-shirts. Max kept updating the diary on the wiki. Ogaga made a miniprep and along with Sam, performed a ligation and transformation. Overnights were made with the E. coliincluding our AMP gene and untransformed supercompetent E. coli cells plated in preparation for our arabinose testing tomorrow. Charlie ordered primers for the Drug Delivery Proof experiment through IDT.

Day 5:

We had colonies from both E. colisubtypes and the arabinose test was carried out by Sam and Lamya. No transformed colonies were detected so another transformation was done by Ogaga which will be checked tomorrow. Max continued to look into MATLAB procedures to input our equations. Sam continued to work on the presentation, adding slides, compiling data and finding sources.

World Dundee Worlds Debating Championships 2011:

The team visited Dundee Worlds Debating Championships 2011. The presentation was finished and rehearsed with Sam presenting. Videos and photographs were taken of both teams. Dundee supported the pros of synthetic biology while St Andrews supported the cons. The event was chaired by Frank Sargeant, and run in part by John Urch. After our presentations, there was an open floor discussion with questions directed towards our student panel. A variety of questions were asked - including whether there might be a cure for AIDS, cancer (through stem cell technology), patenting, and about synthetic biology having an adverse effect (such as soil erosion/desertification/etc.).

Day 1:

Sarah arrived today and the team had their bi-weekly meeting. We ran a gel on the ligation to see the reason why the transformation failed, and found that the ligation itself had failed. We have done PCR on our mini-prep backup DNA. Plates of super-competent E. coli were made as a control for the arabinose test to be redone on Wednesday.

Day 2:

The PCR failed as the tubes opened up twice, which caused one of the tube's DNA to evaporate, however the other tube was saved. We decided not to carry out a digestion on the PCR material, as we ran it on a gel and the results seemed to show that our desired product (AMP gene) was present in ample amounts. Overnights were made for the arabinose test to be done tomorrow. Simple graphs of the experimental results from last Friday were produced and discussed. Max inputted the first three equations into MATLAB and found they were producing a straight line, which the modellers were quite wary of and decided to look more into it. Charlie finished the re-writing of the safety form. Chris continued to look at various statistical tests for the human practices spreadsheet.

Day 3:

Sam worked on a ligation experiment that involved running several ligations for different lengths of time. We ran these ligations on a gel to find the optimum length of time required to run a successful version. A subsequent ligation was then made to run for this optimum time, with the intention to run a transformation the next day. Lamya and Ogaga attempted another Kill Switch Proof experiment. The modelling team looked at changing the order of magnitude of the constants to glean results, looking in particular at the diffusion constant of arabinose into the cell. Five separate graphs imitating what was expected to occur in the cell were produced.

Day 4:

We had our bi-weekly morning meeting with our supervisors. Charlie created a plan covering the next two weeks in an attempt to keep all the lab work on schedule. We discovered we had been using D-arabinose instead of L-arabinose due to an ordering error, which explained why our Kill Switch Proof results had not been very successful so far. The modelling team looked to see the effect of having a log scale on the vertical axis, and how those graphs portrayed multiple parts of the system. They also looked into altering the concentration of arabinose outside the cell will have an effect on the output of protegrin-1. For the human practices review, the team decided that we would no longer use hypotheses to test statistical data; instead we wanted to have a broad spectrum of statistics comparing all the variables against each other. This way will have a more conducive idea of what variables are independent/dependent. A few biology lecturers with a strong knowledge of bio-statistics were e-mailed to help out.

Day 5:

We ran a gel of the ligate and discovered that it had not worked due to the fact that we didn’t digest the DNA for sticky ends! We noted our mistake and decided to repeat the ligation. The modelling team ran simulations to find the best ranges for transcription rates/translation rates/degradation. Charlie and Anne decided that for the Drug Delivery Proof, the "drug" would be his-tagged GFP. The GFP would be big enough that it wouldn't be naturally exported from the cell, and the his-tag would allow us to quantify the amount present in the extracellular matrix post cell death. L-arabinose arrived in the mail.

Day 6:

Charlie transformed the GFP biobrick (I20260), and the terminator (B0015), and the backbone (J04450).

Day 7:

Charlie transformed J04450 again because it failed, and made overnights for the arabinose test to be re-done tomorrow, but this time with L-Arabinose.

Day 1:

The modelling team were rerunning and narrowing/rewriting the parameters to create a more efficient model. The drug proof experiment began. Ogaga made a miniprep, while Sam did a PCR on our GFP-HIS tag plasmid and terminator for the drug delivery proof experiment. PCR also carried out on the Backbone (iGem Biobrick backbone).

Day 2:

The modelling team ran through a variety of variables to see which would be the best set to utilise in our system and have narrowed down translation and degradation for PG-1. Chris talked about to Lorna Sibbett about the statistics involved in our review who then suggested a couple of ideas and even loaned us a textbook. We attempted another Arabinose but an error occurred so we have agreed to carry it out again

Day 3:

The modelling decided upon a translation rate and degradation rate that is suitable for our system. They will, however, continue to alter ranges to find optimum value. We finally carried out a proper arabinose experiment today. The findings were analysed and graphed in excel. The discussions affecting the results included the fact that the cell would use the arabinose addition as food to consume as well as being used in the activation of the promoter. We also discussed a possible equilibrium with ribulose.

Day 4:

We received some PSB1C3 backbone from Glasgow, as our supplies were running low and PCRing some more was becoming problematic. Charlie carried out another run of the Kill Switch Proof experiment after the previous one produced unsatifactory results. Sam carried out PCR on miniprepped J04450 DNA, as well as a digestion and ligation. Overnights were made of the backbone, supercompetent E. coli and our AMP E. coli to obtain repeats of the Kill Switch Proof experiments, as well as make glycerol stocks for storage.

Day 5:

The modelling had a look at differing ranges of the transcription rates and used a logarithmic y-axis to scale all concentrations onto the same graph to see if there’s a ‘switching on/off' behaviour. Chris looked into the stats and read up on trialing the data in basic histograms to explore any underlying correlating behaviours. Max read up on animation software to create something for our wiki.

Day 7:

Charlie made overnights of supercompetent E. coli and E. coli with our AMP gene.

Day 1:

We had our bi-weekly meeting with our advisors. Sam did a PCR and ran a gel of it to check if it worked and it didn’t. We decided to repeat the reaction. Ogaga prepared overnights of supercompetent E. coli and E. coli with our AMP gene as there was an error in the one made the previous day. Max worked on animation of the biological system. Chris went to meet Will Cresswell and had a run through of converting the data from SPSS to CSV to R, modelling the ideal model for our stats. There seems to be no significant p-values for most of the variables. Although when applying a model setup in R, there is no significance between student-advisor ratio and the medals given.

Day 2:

Lamya and Ogaga made minipreps of the J04450 backbone. Sam made a PCR of the minpreps and Charlie ran another arabinose test. Ogaga also streaked a plate for singles of the backbone. A variety of models were run in the stats of the human practices.

Day 3:

Sam ran a gel today of the PCR. Chris ran various models, such as the dependency of projected budget on the team, whether the projected budget has an effect present from the citation score and overall rank of the university. Also the division and year variations, to see if the random effects that have been removed from the model.

Day 4:

We had our bi-weekly meeting with our advisors. Chris submitted modelling essay. For the modelling, the last modelling graph, representing PG-1, shows oscillations into the negative concentrations. This, however, cannot be correct. A variety of methods were deployed to see if MATLAB’s code solver was taking inappropriate time steps at each iteration of the model, such as running the differential equation system in a different mathematical program. Also the model is going to be stopped once PG-1mRNA has reached the limit of 1e-. to see what the values of the other parameters are at this point. Hopefully this should highlight some of the parameter values at that time. Returning to MATLAB, we decided to look at forcing the time-step for the model, to provide a smoother output. For human practices, we decided to provide graphs depicting the difference of the total advisors and the variation in awards. We also attempted to have each year side-by-side, to show the implication of the total advisors between each competition year. We also looked at different experimental methods to prove our biobrick works such methods included microscopy and staining. Sam preformed some more ligation, digestion and PCR.

Day 5:

Sam did a ligation in the lab today and ran a gel on his previous ligation experiments. Essays were put up on our wiki as part of our human practises project.

Day 1:

We had our bi-weekly meeting with our advisors. Ogaga transformed the ligation made on Friday while Sam made another ligation and ordered some more ligase. We ran a gel on the ligation and discovered to our dismay another failure.

Day 2:

We found colonies today on the plates from our transformations but went on to discover that the ligation must have failed because the colonies were pink. Chris had a meeting with Dr Chris Hooley to discuss. We decided to have a new approach (microscopy) to our experimental method as the measurement of absorbency didn’t seem to work. Sam and Charlie went to the medical schools’ histology lab to carry out the experiment. We also ordered Baclight- a stain required to see E.coli under a light microscope because the microscopy experiment failed to yield positive results due our inability to see the cells properly.

Day 3:

Our DNA ligase arrived today. Sam carried out a digestion and ligation today. A lot of work was done on our wiki in preparation for the deadline which is in a weeks’ time.

Day 4:

We had our bi-weekly meeting today. The baclight also arrived today. Sam made a ligation today and the team continued to update the wiki. David, a member of the St-Andrews iGEM team 2010 came over to the lab to help with our ligation. He took the ligation made by Sam as well as a plate with colonies and a miniprep to Dundee, which is where he is based. He performed a digestion on the ligation which yielded very faint bands at the right location on a gel and overnights made of the colonies.

Day 5:

The team continued to update the wiki. Ogaga and Lamya carried out a transformation on supercompetenet cells of the ligation made on Thursday. Charlie performed a new characterisation experiment using baclight and a microplate reader while Sam carried out another ligation and ran a gel on it. By evening, the transformed cells appeared pink indicating RFP. David, still based in Dundee, ran a digestion of the DNA 2.0 synthesis product miniprep (our AMP gene). This was run on a gel and a faint band was found at approx. the right position. This band was cut out and purified. The DNA concentration following the gel extraction was very low (<5ng/ul), but a ligation was put on using the backbone made by Richard, David’s brother a member of the Dundee iGEM team 2011.

Day 6:

Charlie ran another characterisation experiment using baclight and a microplate reader. David transformed the ligated DNA into super competent cells. Sam and David in St Andrews, ran a PCR on the backbone (J04450) and insert (AMP gene) using the iGEM primer design. This was then purified and digested with EcoR1 and Pst1. This was then ligated and transformed into super competent cells.

Day 7:

Charlie ran another characterisation experiment today using baclight and a microplate reader but with a different protocol. David brought the transformed cells he made along with Richard over to St-Andrews. These cells were white indicating that a problem occurred in the PCR, causing it to fail to remove RFP. A colony PCR was carried out on the successfully transformed cells. This was run on a gel and bands were seen at 370bp which is about the size of our insert. Overnights of these colonies were made.

Day 1:

We had our final advisors' meeting. The team continued to update the wiki. A miniprep was made of the overnights which was then eluted using a TE buffer (to be sent to MIT) and water (for further investigation). Sam also carried out a digestion on the DNA to additionally confirm that the correct DNA was being sent. Overnights were also made for a final characterisation data experiment, involving controls, to be done.

Day 2:

DNA was sent to the registry in the morning. Another repeat of the arabinose experiment was carried out by Charlie. The team concentrated on finishing the wiki in time for the deadline in two days. Overnights were made so that more characterisation data could be obtained.

Day 3:

Ogaga carried out a final run of the arabinose characterisation experiment. The team continued to focus on updating the wiki and collating all the project's data to upload.