Team:NRP-UEA-Norwich/Results
Results
Cloning Results
Cloning the FeFe and NiFe hydrogenase genes individually into iGEM BioBricks, which can then be cloned into pBAD vector using Golden Gate Cloning (GGC).
Protein Modelling
As the native structures of MtrCAB and hydrogenases in Shewanella oneidensis have not yet been resolved. We decided to model these protein structures using homology sequencing. These models would then be used for our Virtual Reality model.
BioBrick Conjugation into Shewanella oneidensis
In order to assess whether our BioBricks would function in our chassis organism Shewanella oneidensis MR-1 we attempted to transform and express two BioBricks into S. oneidensis MR-1; J04450 containing a gene for the red fluorescent protein (RFP) and K584001 which contains a gene for the green fluorescent protein (GFP). These BioBricks were chosen as they contain reporter genes and successfully transformed colonies could be easily identified under UV light.
Triparental conjugation and electroporation of plasmids into Shewanella oneidensis
Attempted a triparental conjugation and additional electroporation of plasmids containing HydABC Strep-II tag on C-terminus and HydABC Strep-II tag on N-terminus constructs into wild type, double knock out and FeFe knock out Shewanella oneidensis MR-1
Anaerobic growth of Shewanella oneidensis MR-1 to catalyse H2 evolution
The electrochemistry aspect of our project involved anaerobically growing up both the Wildtype (MR-1) and ∆HydABC,HyaABC Shewanella oneidensis strains. After which, we quantified the amount of hydrogen in the headspace gas using gas chromatography.
Protein expression assays for 8A5 golden gate construct of FeFe hydrogenase gene cluster into the pBAD vector
Cloning the FeFe and NiFe hydrogenase genes individually into iGEM BioBricks, which can then be cloned into pBAD vector using Golden Gate Cloning (GGC).
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