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Team:St Andrews/diary
From 2011.igem.org
Diary
Pre-Summer
Meeting 1
On our very first meeting, we had a brain storm about possible projects. The first ideas can be seen on the mind map below.
Meeting 2
On our second meeting we tried to refine the ideas and come up with more possible projects.
Meeting 3
After doing some further reading, we narrowed some of the topics down.
Meeting 4
At this meeting we discussed the possibility of our team participating at the St. Andrews annual outreach event during the NSEW (National Science and Engineering Week) between the 11th and 20th of March.
“National Science & Engineering Week shines the spotlight each March on how the sciences and engineering relate to our everyday lives and helps to inspire the next generation of scientists with fun and participative activities.” from British Science Association
The plan was to tell children about the functions of specific sections of DNA – promoters, ribosome binding sites etc., and create a simple game which could enhance their understanding of genetics. We decided on using mega-blocks of different shapes to represent a promoter, a ribosome binding site, a gene and a terminator and allow the children to put the pieces together in the correct order to make a “bio-brick”.
Meeting 5
For the NSEW event we decided to use a poster and instructional cards to aid in our ability to help children understand the functions of different segments of DNA. After a lot of deliberation, we came up with the following poster.
Meeting 6
At this meeting we discussed the idea of using apoptosis as a means of drug delivery and thus using the cell as a factory for drug creation. We realized, however, that the destruction of the cell's organelles and components upon apoptosis may lead to the destruction of the drug. We then discussed other means of transport of the drug such as a pump that could release the drug molecules when it encounters an environmental trigger such as pH.
We also discussed contacting members from last years’ St. Andrews iGEM team to familiarize ouselves with various laboratory procedures that we would use over the summer.
For our next meeting we decided to do some research into whether the pump we required existed and what restrictions it would place on the drug's molecular structure.
Meeting 7
We discussed the possibility of modifying our project -"Drug Delivery to the Gut using bacteria" to make the bacteria produce another drug or a protein which could potentially treat or cure another disease.
We also considered exploring prokaryotic cell death signals as apoptosis is not very feasible in prokaryotic cells like bacteria.
Meeting 8
In our last meeting before summer we explored the possibility of making bacteria secrete other substances in the gut which could enhance immunity – or perhaps create a kill switch in the bacteria which could have various applications in drug delivery. At this point we explored the functions of anti-microbial peptides and how we could use them to create a kill switch.
We did some research into antimicrobial peptides and decided that our project would be to create a ‘kill switch’ where we would insert a gene (protegrin-1) coding for Anti-Microbial Peptides (AMP’s) into E. Coli. The idea was to then use different promoters to switch on the expression of these proteins in certain conditions
Summer
Week 1 - 11/07/11
Day 1: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 divided 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 learnt 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.
Day 2:
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.
To finalise the lab team prepared overnights of E. coli in Luria broth so that they could run a practice transformation and gel electrophoresis later in the week.
Day 3:
We completed the Project Summary form and Safety Proposal and put them on the team wiki, the team then concentrated in finishing with the pre-summer meeting material. After completing the sponsorship proposal Christina began emailing potential sponsors.
In the meantime the lab team made the E. coli from the overnights and carried out practice transformation experiments using a biobrick which provides resistance to ampicillin.
Day 4:
Charlie and Ogaga began researching various constitutive and positive promoters, in order to complete our basic biobrick sequence and order DNA.
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.
Day 5:
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, etc.
Week 2 - 18/07/11
Day 1:The lab team re-did the gel electrophoresis which had previously shown no presence of DNA, with a smaller gap of time 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 Anne ordered the necessary DNA.
Christina and Lamya continued emailing and calling sponsors while Max continued working on the team wiki.
Day 2:
The team explored conjugation and drug-delivery as potential applications of our project and we explored the IGEM registry for biobricks that could help us test the feasibility of our applications.
Later on we brainstormed ideas for our human practices project and discussed some potential questions that could be addressed.
Day 3:
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 university over the world.
Sam, Ogaga and Lamya collated the data for all the 2010 human practices projects and concluded that the projects were too divergent to show any significant trends that could be used to draw conclussions. Leading us to abandon the idea of using this idea as our human practices project.
Day 4:
We prepared and practiced the presentation we planned to give at the UK IGEM Meet-up in Norwich on Monday while Max continued working on the wiki. Charlie and Ogaga concentrated in designing some experiments and controls to test the potential applications of our project.
Day 5:
The team discussed further on potential ideas for the human practices component of our project and we agreed on a brilliant and inovative project. This project however required support from organisers from external events, so we drew a clear HP plan and contacted the relevant parties.
Week 3 - 25/07/11
Day 1 and 2:
The St. Andrews IGEM team flew to Norwich to participate in the UK IGEM meet-up where we presented our project idea and progress to date. At Norwich, we were able to interact with other IGEM teams 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 began this day at 1pm and spent time discussing about our time in Norwich, the lessons learnt and their possible applications in relation to our project.
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 contacted the World debate championships in Dundee to confirm our place at the debate.
Day 5:
The team made plans for the debate and focused on planning the experiments for our drug proof and checking the viability of conjugation based on our available time and team size. A lot of research was also done into conjugation.
Week 4 - 1/08/11
Day 1:
The team had a morning meeting with the advisors present meanwhile, Charlie met with Dr Coote an expert in molecular Biology. We brainstormed some ore thoughts on our human practises focusing the the idea of a review. At the end of this we came up with a number of variables and began extracting data from the wikis of previous iGEM teams.
Day 2:
The team focused mainly on the extraction of data today. We all spent time researching online to find various figures for our iGEM review.
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 set up a meeting for week. 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, 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 from iGEM as well as our Biobrick in the post, Charlie tested SDS(lysing agent) absorbance and found a result that made us rethink our experiment. Modelling team continued to read papers while Ogaga, Lamya and Sam continued gathering information for our Human Practises component.
Week 5 - 8/08/11
Day 1:
We had our Bi-weekly meeting. The modelling Team read papers and found a few regarding the sigma 70 factor. They emailed John about pBAD advice and transcription rates. The Human Practises team continued gathering information. We also obtained DNA from the filter paper that we received it.
Day 2:
We transformed supercompetent E Coli by placing our plasmid in it. The modelling team tried to create a hybrid system of equations from last year and Berkeley 2008. At the end of the day they were still left with a lot of unknown constants that have been trying to source from papers.
Day 3:
Sam, Max and Charlie visited Dundee to make plans concerning the debate collaboration. Ogaga made overnights. The Human Practises team continued gathering information.
Day 4:
We had our bi-weekly meeting with our advisors today. The modelling team looked into finding the right constants. Sam and Charlie did a run through of the arabinose experiment, which proved to be fruitless.
Day 5:
We got further response from sponsors. Russell (BioSilta) gave us a voucher worth £500 so we could order anything to that value from their catalouge. The modelling team met with Chris Hooley and decided that their system is basic enough. They also concluded that adding another set of equations relating the pBAD repressor (AraC) and pBAD promoter concentrations will be needed. They however still have to fine 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 the PG-1 production.
Week 6 - 15/07/11
Day 1:
We had our Bi-weekly meeting with our supervisors. We responded to Russel from Biosilta with a list of the things we may need in the lab. 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, a contact obtained from Sarah. The database was also completed today, a huge success for the Human practises 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, Arabinose+AraC complex concentration linked to the promoter ‘strength’ and therefore the transcription rate. Dr Roger Griffiths, a specialist in enzyme kinetics, was emailed to see if he could help out. Ogaga and Sam performed a miniprep and digestion while Charlie ordered primers. The team also worked on the Dundee presentation and started collating quotes and ideas for the cons of synthetic biology, to present to the students at the World schools debating championship.
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 today. Max kept updating the diary on the wiki. Ogaga made a miniprep today and along with Sam, performed a ligation and transformation. Overnights were made the E. coli with our AMP gene and 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 obtained colonies from supercompetent cells and the arabinose test was carried out by Sam and Lamya. We added arabinose to see if the AMP gene works as expected. No transformations 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 nad finding sources.
Day 7 World Dundee Worlds Debating Championships 2011:
The team visited Dundee Worlds Debating Championships 2011. The presentation was finished and rehearsed with Sam presenting. Videos (st Andrews) and photographs were taken (Dundee) of both teams. Dundee supported the pros of synthetic biology while St Andrews supported the cons. e event was chaired by Frank Sargeant. There was an open floor discussion to a panel of students. A variety of questions asked - including whether there might be a cure for AIDS, Cancer (through stem cell technology), patenting, synthetic biology having an adverse effect (such as soil erosion/desertification).
Week 7 - 22/08/11
Day 1:
Sarah arrived today and the team had it bi-weekly meeting. We ran a gel on the ligation to see the reason why the transformation failed. We ran a gel to see where the experiment went wrong.from the gel we saw that the experiment went wrong in the ligation phase. we have done PCR on our mini-prep backup DNA. Plates of super-competent Ecoli were made as a control for the arabinose test to be redone on Wednesday.
Day 2:
The PCR failed as the tops of the tubes opened up twice, one tube was however saved. The PCR finished today. We decided not to carry out digestion on the PCR material, as we ran it on a gel and the results seem to show that our desired product (AMP gene) is present in ample amounts. Overnights to were made for the arabinose test to be done tomorrow. Simple Graphs of the experimental results from last Friday were produced and discussed. The Modelling team inputted their first three equations into MATLAB and found they were producing a straight line which they were quite unsure of and decided to look more into it. Charlie finished the safety form. Chris compared variabes to apply stats in the HP spreadsheet.
Day 3:
Sam worked on the ligation experiment, while Lamya and Ogaga worked on the arabinose experiment today. We ran the ligation on a gel to see the optimum time to leave the ligation for. Another ligation was then made at the optimum time and a transformation will be carried out tomorrow. The modelling team looked at changing the order of magnitude of the constants to glean results, especially the diffusion constant of arabinose into the cell. They had five separate graphs imitating what we expect to see occur in the cell.
Day 4:
We had our Bi-weekly morning meeting with our supervisors. Charlie created a lab schedule covering the next two weeks. We discovered we had been using D arabinose insead of L Arabinose which explained why our results had not been very good so far. The modelling team looked to see the effect having a log scale on the vertical axis on the ability of the graph to show all parts of the cell. They also looked into altering the concentration of arabinose outside the cell will have an effect on the output of PG-1. (Altering ranges). For the review the team decided that we would no longer use hypotheses 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’s independent/dependent. A few biology lecturers with good 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 probably 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 and ultimately the input arabinose concentration as these values are the least well known with regard to the pBAD promoter.
Day 6:
Charlie transformed the GFP biobrick, I20260 and the terminator, B0015 and the backbone, J04450.
Day 7:
Charlie transformed the backbone again because it failed and made overnights for the arabinose test to be re-done tomorrow but this time with L-Arabinose.
Week 8 - 29/08/11
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 backbone from Glasgow. Charlie carried out another run of the arabinose experiment after the previous one produced unsatifactory results. Sam carried out PCR on the backbone as well as digestion and ligation. Overnights were made of the backbone, supercompetent ecoli and our AMP ecoli to obtain repeats of the arabinose experiments as well as make glycerol stocks for storage.
Day 5:
The modelling had a look at differing ranges of the transcription ranges and used a logarithmic y-axis to scale all concentrations onto the same graph to see if there’s a ‘switching’ on 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 aniation software.
Day 7:
Charlie made overnights of supercompetent e coli and e coli with our AMP gene.
Week 9 - 05/09/11
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.
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Week 10 - 12/09/11
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