Week 1—Commencing 23rd June

Aims: This week our main aim was to provide everyone with a basic understanding of synthetic biology, due to many of our team members having a limited biological background. This was comprised of some talks from our advisor as well as practice sessions in the laboratory in order to familiarise our selves with the basic techniques that would be needed throughout the project.

23/6/14 Today comprised of background talks by our supervisor, outlining the basics of DNA replication; transcription; translation. We also had a discussion about basic process of cell growth and the phases that are involved. We also completed the about our lab form.

24/6/14 Today Randy Redford came to the university to talk to us about IGEM and to give us some advice on how to proceed with the project. Randy also gave a lecture on synthetic biology and how it could be used in the future.

25/6/14 Today was spent researching into previous teams projects. This involved looking into the previous winners projects; their wiki’s and their posters to see what was required for a winning project.

26/6/14 Today we made competent TOP10 cells (see the attached protocol). These are the cells we used in the future experiments.

27/6/14 Today we transformed (See the attached protocol) some of the competent cells with DNA from the registry to confirm the competency of our cells. This also taught us the basic technique of Transformation.

Week 2—Commencing 30th June

Aims: This weeks main aim was to begin investigating into potential ideas for our project. This involved investigating the feasibility and originality of our ideas. To do this we arranged meetings with specialists and academics in the field. On the Friday we arranged to have a meeting with Warwick ventures; a panel of business experts who offered their view on the business side of the potential ideas; this was done in a dragons den style.

30/6/14—1/7/14 Today we began by mind mapping potential ideas that could be investigated. These ideas were the divided between the group. The initial set of ideas that we investigated were: Carbon capture; urine cancer test; parasite detection; polymer production e.g skin/bone; metal recovery; bee diseases; fortified food; alcohol sensors; methane capture; Tyrian purple and miRNAs.

2/7/14 Today we looked through all of the research we had collated on each of the ideas in order to dismiss and narrow down our ideas into which ideas we should present at the dragons den session on the Friday. The final ideas we decided on were: the production of Tyrian purple; Nano compartments; methane capture.

3/7/14 Today we planned out our pitches for Warwick vetures. This involved arranging meetings with Tim Bugg to discuss the feasibility of converting the chemical process of tyrian purple production into a biological process. As well as this we discussed the uses and feasibility of generating Nano compartments.

4/7/14 Today was the meeting with Warwick Ventures where they gave their business point of view on our ideas. They also gave us some general advice on how to pick a project and how to run it successfully

Week 3—Commencing 7th July

Aims: The aims of this week were to select the main aim of our project and to identify which elements would be needed in order to carry out our project.

7/7/14 Today we discussed and weighed up all of the ideas that we had been considering as possible projects. From this we decided to focus our project on miRNAs / siRNA therapy.

8/7/14 Today we investigated different directions we could go with siRNAs. The directions that we decided to investigate were the use of siRNA for dengue; Alzheimer's; Obesity; diabetes; HIV, cancer; Hepatitis C and cardiovascular disease.

9/7/14 Following our research we settled on the idea of using siRNA to target diabetes. However following our research we decided that we should make a self replicating system based on the hepatitis C virus using the same RNA dependent RNA polymerase (RdRp) the Hepatitis C virus uses.

10/7/14 As we decided to use a self replicating system there needed to be regulatory aspects in the system. We decided to introduce an immediate kill switch the and a long term less extreme regulation e.g a feedback mechanism.

11/7/14 We also decided that as the system relies on a functioning 3’UTR we would have to find many options for this. We also had to find a way of cleaving the terminator and the promoter from the RNA following transcription; as this could interfere with the functioning of the system.

Week 4/5—commencing 14th July

Aims: The aims of these weeks were to find the sequences for the requisite parts of the replicon.
Week 4 This week was involved finding the sequences for all of the elements of our project. The sequences were obtained from various pieces of scientific literature (see part description). The sequences then had to be cross referenced to ensure that they were correct and would work in the human and E.coli cell lines. This week we also modified the sequences that we had obtained so that they were optimised for our system this included codon optimisation and the insertion of stop codons. The final thing that was achieved this week was the design of the siRNA section of the replicon, this was design that so when it was in the positive sense the siRNA was inactive and therefore would not attract dicer to it, preventing the targeted destruction of our system. However in the negative sense it would be active and target the RNA required.

Week 5 This week mainly involved the putting together of the sequences. When putting together the sequences we also had to consider the way in which we would test certain elements of our replicon, this required the creation of testing modules. Once we had the order of the parts and the testing modules we had to add into our design restriction sites so that we could swap out some of the parts, we also had to make certain modifications to the replicon to make it bio-brick compatible. The final achievement for this week was to send the modules for synthesis, this required the splitting if the replicon into 3 pieces with each piece being below 2000bp. Whilst sending the parts for sequencing we also had to add to the modules homologous regions for the splice protocol.

Week 6—Commencing 28th July

Aims: The aims of this week were to design in detail our experimental protocols, whilst waiting for the sequencing to return.. This week we also ordered all of the reagents that we would need for the experiments

Week 6 This week we took our rough outline of the experiments and defined them more clearly. This involved outlining exactly what we would have to do to test each part of the replicon as well as the positive and negative controls that would be needed. As well as this we created a timeline of when we would like to have dine all the experiments by and in which order they would need doing. Finally we ordered all the reagents we would need for the experiments, many of which we got free from our sponsors.

Week 7/8—commencing 4th August

Aims: The aims of these weeks were to clone in all of the parts (G blocks ) into the C3 plasmid.
Weeks 7/8 As the parts were of varying sizes they arrived at various points throughout these two weeks. As the parts arrived we had to rehydrate the G block, ligate some of the with linearized plasmid via restriction digestion and then transform cells and plate them. From this overnight cultures were made and then mini-prepped and sent for sequencing. On some of the larger modules this did not work and so the remaining G block had to be PCR amplified prior to the ligation in order to amplify the DNA concentration. Once the parts had been successfully cloned into plasmids and sequenced we were able to make glycerol stocks and begin the testing of the parts.

Week 9/10—Commencing 18th August

Aims: The aims of these week were to clone in the parts for testing into the testing modules as well as begin preparing for slice.
Week 9/10 These weeks involved cloning the IRES’s into the forwards GFP ; the 3’UTRs into the reverse GFP for human and E.coli cells; the aptazyme and the siRNA target into the forwards GFP testing module. Following this the modules were then sequenced so as to confirm that the DNA in the plasmid was what were expecting.

Week 11—Commencing 1st September

Aims: This week the aim was to test the 3’ UTRs in E.coli cells.
Week 11 This weeks involved transforming the plasmids into cells with a T7 promotor. These then had the media refreshed and were induced using IPTG. The E.coli samples were run in the Tecan a device that measure fluorescence over time.

Week 12—commencing 8th September

Aims: The aims of this week were to test the 3’UTRs in human cells
Week 12 This week involved invitro transcription to RNA of the composite plasmids. The human cells were then transfected with the RNA. The media had to be refreshed and were induced using IPTG. The samples were run in the Tecan. These were done separately to the E.coli samples to prevent contamination.

Week 13—Commencing 15th September

Aims: The aims of this week were to test the IRESes
Week 13 This week involved invitro transcription to RNA of the composite plasmids. The human cells were then transfected with the RNA. The media had to be refreshed and were induced using IPTG. The samples were run in the Tecan.

Week 14—commencing 22nd September

Aims: The aims of this week was to test the aptazyme
Week 14 This week involved invitro transcription to RNA of the composite plasmids. The human cells were then transfected with the RNA. The media had to be refreshed and were induced using IPTG. Some of the samples were treated with varying concentrations of theophylline to access the efficiency of the aptazyme The samples were then un in the Tecan.

Week 15—Commencing 29th September

Aims: The aims of this week were to characterise a part from the registry
Week 15 This weeks involved transforming the plasmids into cells with a T7 promotor. These then had the media refreshed and were induced using IPTG. The E.coli samples were run in the Tecan.

Week 16—commencing 6th October

Aims: The aims of this week was to repeat any experiments that did not work properly or needed more replicates.
Week 16 This week we repeated some of the experiments such as the IRES experiments in order to get more replicates and some cleaner results.