Vilnius

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 the completion of the modelling for the team. We have created models to simulate the conversion of phenylalanine into trans-cinnamic acid by phenylalanine ammonia lyase (PAL). To do this we used MATLAB to solve ordinary differential equations that predict the expression of the enzyme and rate of the reaction over time. We have provided them with the graphs, equations, and information on their wiki page.

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.

Cardiff

We discovered a innovative paper by Hotzer et al⁽³⁾ that described how His-tagged GFP can be quenched by a copper ion binding to this His tag leading to a reduction in the fluorescence lifetime. They speculated that this could potentially be used as a in vivo copper assay.

As we had His-tagged our chelator-sfGFP constructs we were curious to see if this technique could be applied to our parts to measure copper chelation in vivo by our parts. We believe that two possibilities were likely:

1. Copper chelation by the chelator reduces the free copper concentration inside the cell meaning that less binds to the His tag and the fluorescence lifetime will be greater than a His-tagged sfGFP control
2. Copper chelation by the chelator would allow additional quenching if copper was bound within the quenching radius of the fluorophore leading to a reduction in fluorescence lifetime compare with a sfGFP control

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.

As expected the pCopA CueR sfGFP control was fluorescent, with and without arabinose, with the mean fluorescence lifetime a consistent 2.6ns.

When Csp1-sfGFP was induced the mean lifetime decreased to 2.5ns and the variance was increased. This might be indicate that copper chelation has occurred or may be reflective of the expression problems of Csp1.

When MymT-sfGFP was induced the mean lifetime decreased to 2.3ns. As MymT-sfGFP is was observed to be reliably expressed and because MymT is a small copper cluster separated form sfGFP by a small linker we believe that this represents additional question of the fluorphore by MymT-bound copper showing in vivo copper chelation.

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 differ overnight copper concentrations.

Our most sincere gratitude to all those at Cardiff iGEM in particular Rob Newman for setting up the collaboration, Geraint Parry for setting up the experiment and Anthony Hayes at the bioimaging unit measuring the our parts using the FLIM microscope.

UNBC

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.

XMU China

We sent team XMU-China some of the parts 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.

SVCE Chennai

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.

Illinois

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 proteins expression issues. As we both were delayed in getting our part constructs prepared we ran out of time to carry this out fully.