Team:NRP-UEA-Norwich/Triparental
RESULTS
To transform the plasmids containing C-terminally strep-tagged & N-terminally strep-tagged hydABC FeFe hydrogenase genes into wild type Shewanella oneidensis MR-1 (WT), S. oneidensis with both hydrogenase genes knocked out (ΔHydABC,HyaABC) and S. oneidensis with the FeFe hydrogenase knocked out (∆HydABC). The pBAD vector was used as the previous experiments attempting to transform BioBricks into S. oneidensis failed (see BioBrick conjugation ).
The tri-parental conjugation protocol was initially carried out, as S. oneidensis MR-1 are not susceptible to heat shock. We used strains of Escherichia coli containing the plasmids generated by this experiment; see here. This was attempted twice but yielded no results, so electroporation was used as an alternative method. The protocol was followed for the electroporation; see here . To check that the plasmid was in these strains, colonies were inoculated from each of the plates and then a Miniprep was performed according to the protocol. Agarose gel electrophoresis was run with the original hydABC C-terminally tagged and hydABC N-terminally tagged plasmids along with the Miniprep DNA. The DNA was also sent off for Sanger sequencing.
Triparental conjugation was attempted twice, but each time no colonies of S. oneidensis were observed when plated out onto kanamycin selection plates for any of the combinations of plasmid and strain type. Electroporation was performed as another method to try to get both plasmids into the three different S. oneidensis strains (Wildtype MR-1, Double hydrogenase knock out ∆HydABC,HyaABC and FeFe hydrogenase knockout ∆hydABC. On the second attempt, results from the DNA sequencing confirmed that hydABC N-terminally SII tagged had been transformed into wild type S.oneidensis. This gave us a cell line of MR-1 containing an arabinose inducible hydABC gene to be used in following proof of concept experiments (from this point denoted MR-1:HydABC)
Figure 1: Kanamycin plates showing successfully transformed colonies Wild Type Shewanella oneidensis MR-1 containing the golden gate construct . This construct contains the hydABC gene cluster, with a C-terminal strep II tag.
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