Team:NRP-UEA-Norwich/Collaborations/kent
Collaborations
We have collaborated with UKC to image the adherence of E. coli and S.oneidensis MR-1 onto graphite and iron. In return we offered to model their proteins. This collaboration gave us a better indication of which possible cathode materials could be used in our electrochemical system when we have prepared our modified strains of Shewanella oneidensis MR-1.
At the iGEM Westminster Meetup we got into contact with the iGEM Kent team (UKC) and started discussing potential ideas for collaboration. We established an experiment which would address the adherence of S. oneidensis MR-1 onto potential electrode materials. UKC would provide us access to their Atomic Force Microscopy (AFM), and in return we would model their proteins.
Figure 1: Atomic Force Microscopy (AFM) of S.oneidensis MR-1 on graphite (Figure 1A) and iron (Figure 1B) after 12 hours incubation. (A) S.oneidensis MR-1 adhered to the proposed cathode material, graphite, after incubation time of 12h in LB broth. Graphite discs were sculpted from 9B graphite pencil and glued onto 10mm Mica Iron discs. (B) S.oneidensis MR-1 did not adhere to iron after incubation time of 12h in LB broth, which acted as our positive result. Iron discs were Mica Iron 10mm discs purchased from Agar Scientific.
The AFM results showed that S.oneidensis MR-1 could adhere to graphite, figure 1A. Unfortunately, no S.oneidensis MR-1 adhered to iron which should have been the positive control, figure 1B. After discussions with Kent we determined that this issue could be due to the treatment of the Iron Mica discs used as sample slides for the atomic force microscopy i.e. it could be coated with a material that S.oneidensis could not adhere to. If we were to repeat the experiment we would use untreated iron or manganese as a positive control.
We modelled UKC’s native and non-native, proteins using the Phyre 2 server (see figure 2). The protein models showed low alignment, high disorder and low sequence coverage, this is due to the fact there are few proteins with high sequence homology that have crystal structures resolved for the proteins UKC were working with. The models we produced were the best possible approximation of these proteins structures from homology based modelling. Thus, giving our attempt at modelling their structures gave them a rough indication of what their proteins could look like.
Figure 2: Mam X soluble cleaved protein model . Intensive search of UKC’s Mam X soluble-cleaved protein on Phyre2 to try model a 3D structure of the proteins. The intent behind this modelling aspect of the collaboration was to use our knowledge of homology protein modelling to help UKC approximate the structures their protein models after they attempted to remove the transmembrane region. Simultaneously, they helped us in observing the adherence of S. oneidensis to potential cathode materials. The results on both ends were not what was expected in that while the bacteria did adhere to the graphite they did not adhere to iron, and we did not manage to take these experiments further to start optimising biofilm growth conditions. We demonstrated however that S. oneidensis will adhere to graphite, and this informed our demonstration experiment. Similarly, for UKC we have attempted to model their protein structures to the best possible approximation.
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