The Vilnius-Lithuania iGEM team are creating a therapeutic probiotic for the treatment of phenylketonuria, similar to our therapeutic for Wilson's disease. We have maintained contact with the team for much of the duration of our project, through Skype and email. The main part of our collaboration with them has been to sort out the modelling for the team.
We agreed to have the modelling of the Vilnius team outsourced to our dry lab team. We created models to simulate their team project, which is to convert phenylalanine into trans-cinnamic acid (TCA) using a recombinant enzyme PAL (phenylalanine ammonia lyase) in vivo. The purpose of the model is to define the efficiency of the system and the limits of its performance in order to show the liability of the approach.
In the initial modelling before the experiment, our model simulated the transport of phenylalanine into the cell and the conversion rate into TCA. Under lack of information on the concentration function of PAL and the transporter protein PheP, we provided simulations for a range of concentration conditions for the two proteins. In later models, we incorporated the PAL expression function gained from experimental data to create a more accurate version of the model. From experimental data of the production of TCA, we were able to predict the concentration of PheP in the cell membrane.
Using MATLAB for simulation, we have provided the Vilnius team with graphs, equations, and information of the model to help them create their dry lab wiki page. Below are some examples of the graphs we have provided:
Further to this, we carried out a joint survey with them. Participants from both of our respective countries filled this out, allowing us to find the public's preferred method of therapeutic delivery and opinion on probiotic use. This cross-cultural survey gave us an idea of how the attitudes to probiotics can vary, and the issues we might face if we were to further develop our project and create a treatment that would be used worldwide.
We wanted to analyse our sfGFP-tagged chelators using Fluorescence Lifetime IMaging (FLIM).
Lacking access to a fluorescence lifetime microscope ourselves we contacted Cardiff iGEM who had access to a FLIM machine in Cardiff's bioimaging unit. They very kindly agreed to run a few samples for us taking up over five hours of microscope time.
We sent Cardiff iGEM our parts Csp1-sfGFP, MymT-sfGFP (both in pBAD) and pCopA CueR sfGFP (as a control) in live MG1655 E. coli in agar tubes. Cardiff grew them overnight in 5ml of LB with 5uM copper with and without 2mM arabinose.
The imaging unit spread each strain on slides and measured the fluorescence lifetime of three areas on each slide. For full analysis of the results see our results page.
From this preliminary data we believe that measuring fluorescence lifetime of a His tagged GFP can be used as a reliable method for measuring copper concentration in vivo and that MymT displays copper chelation activity in E. coli. If we could perform additional experiment we would measure the fluorescence lifetime with different overnight copper concentrations to try to generate a standard curve for quantitative analysis.
Our most sincere gratitude to all those at Cardiff iGEM in particular Rob Newman for setting up the collaboration, Dr. Geraint Parry for setting up the experiment and Dr. Anthony Hayes at the bioimaging unit measuring the our parts using the FLIM microscope.
We discovered that UMBC were investigating a similar project idea towards the end of our project and decided to Skype them to share our experiences of performing copper assays in particular the difficulties we had in optimising our BCS absorbance assay and the choice of reducing agent.
We sent team XMU-China some of the parts (DspB (BBa_K1659200), DspBx (BBa_K1659210), DsbA-DspB (BBa_K165920), DsbA-DspBx (BBa_K1659211)) made by last year's Oxford iGEM team that they were unable to obtain from the registry themselves. This enabled them to carry out their project successfully as they were able to use all the parts that they wanted. We transformed all four parts from storage into the E. coli DH5α strain before mini-prepping, nano dropping and posting to them. This was repeated when the first package did not arrive.
We filled in team SVCE Chennai's survey on lab techniques to pass on our experiences of protocols that worked, and others which didn't, in order to help them improve efficiency for other iGEM teams in the future.
We were in regular contact with Illinois UIUC throughout the project. We planned to test their promoter systems in our flow cytometer to provide a measure of cell variance for them and they would try to integrate our copper chelator Csp1sfGFP with their promoter system to see if that would create more even expression and maybe reduce the protein's expression issues. As we both were delayed in getting our part constructs prepared we ran out of time to carry this out fully.