Cloning the FeFe and NiFe hydrogenase genes individually into iGEM BioBricks, which can then be cloned into pBAD vector using Golden Gate Cloning (GGC).
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
Shewanella oneidensis MR-1 growth to catalyse H2 evolution and demonstration of bioreactor system for electrochemical hydrogen production.
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. We then performed a series of experiments to demonstrate our working electrochemical system in simulated laboratory conditions.
Protein expression assays for FeFe hydrogenase expression from the golden gate hydABC gene cluster construct
SDS page and western blot analysis attempting to confirm expression of the FeFe hydrogenase from our golden gate construct. These assays were testing for protein product produced from our gene cluster via the attached strep-tag