Team:NRP-UEA-Norwich/Results/ProteinExpressions
RESULTS
After successful electroporation< of the hydABC cluster (FeFe hydrogenase expression vector) into Shewanella oneidensis MR-1 SDS page and western blot analysis were conducted to investigate whether the gene cluster was expressing protein. We also wanted to assess the extent to which the arabinose inducible promoter gave control over levels of hydrogenase enzymes present. Our biobricks contain a version of the HydB transfer subunit of the FeFe HydABC hydrogenase enzyme with a C-terminal streptavidin tag which was used as a target for the primary antibody during western blot analysis.
See relevant protocols for S. oneidensis sample preparation, SDS page and western blots. The OD was checked every 2h before the correct OD was reached for induction. The induction phase was done roughly 12h before the cells were pelleted and the appropriate protocol was applied, the pelleted cells were not frozen.
Figure shows an SDS page gel of Shewanella oneidensis MR-1 whole cell lysates from the MR-1:hydABC strain in lanes 2, 3 & 4 and from the ΔhydABC, hyaABC strains in lanes 6, 7 and 8. Comparing lanes 2, 3 and 4 the MR-1:hydABC strain did not appear to show increasing levels of expression from 1mM of arabinose sample (lane 3) and the 10mM arabinose sample (lane 4). Even comparing these to lane 2, where no arabinose was added, there is no discernible difference in the amount of protein expressed. Comparing lanes 6, 7 and 8 the increasing arabinose expression did not appear to make a difference to the levels of protein expressed in the cells. This makes sense as the ΔHydABC, HyaABC strain does not contain our hydABC expression vector and therefore arabinose should have no effect on protein expression. As for lanes 2, 3 and 4, the lack of difference in protein expression levels indicates arabinose may not have induced expression levels of protein above the background levels of expression in the wild type S. oneidensis. This would be clearer but during the electroporation experiments we failed to transform the hydABC construct into the ΔHydABC, HyaABC strain. As the wild type expression background is present in our S. oneidensis strain containing the hydABC expression vector it is difficult to perceive differences in levels of expression.
Figure 1. SDS-page gel of Shewanella oneidensis MR-1 cell lysates with various concentrations of arabinose added at an optical density (OD) of ~0.5nm. Lane 1 contains the size marker ladder, with the size of each fragment in kDa marked to the left. Lanes 2, 3 & 4 contain S. oneidensis MR-1 transformed with HydABC FeFe plasmid (MR1:hydABC). In lane 2 0mM of arabinose was added, 1mM in lane 3 and 10mM in lane 4. Lane 5 is empty. Lanes 6, 7 & 8 contain the double knockout strain of S. oneidensis MR-1 (MR-1:DK), lacking both the FeFe & NiFe hydrogenase. In lane 6 0mM of arabinose was added, 1mM in lane 7 and 10mM in lane 8.
We ran western blots against the strep-tag attached to the C-terminus of the HydB subunit. These experiments failed to confirm expression of the hydrogenase enzymes from our golden gate expression vector. There are numerous possible reasons for this, the obvious one being the proteins weren’t expressing and the construct does not work. However we cannot write off our construct outright based on this data; other researchers in our lab working with the same strep-tag in S. oneidensis also came across issues getting the antibodies to bind the tag. The hypothesis is that the tag gets cleaved off at some point during protein assembly. Due to the short time frame of this project we were unable to explore the issue further. This means our construct may be expressing, a hypothesis supported by the results observed in the electrochemistry experiments.
In the future to confirm arabinose induced expression of the hydrogenase enzymes from this construct we would firstly like to transform the vector into the MR-1:DK strain. This would allow for a clearer picture of different expression patterns to be obtained from SDS page analysis. Additionally, we would generate specific antibodies against the hydrogenase enzymes themselves, in order to eliminate doubts surrounding the strep-tag and confirm that our construct does indeed express our hydrogenase enzymes. Due to time constraints however we were unable to explore these options further.
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