Team:NRP-UEA-Norwich/Triparental
RESULTS
To transform the plasmids containing C-terminally strep-tagged & N-terminally strep-tagged hyaABC FeFe hydrogenase genes into wild type Shewanella oneidensis MR1 (WT), S. oneidensis with both hydrogenase genes knocked out (ΔhydABC,hyaABC) and S. oneidensis with the FeFe hydrogenase knocked out (FeFe knock out). The pBAD vector was used as the previous experiments attempting to transform Biobricks into S. oneidensis failed (see biobrick conjgantion results page).
The tri-parental conjugation protocol was initially carried out, as S. oneidensis MR1 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; see here. Agarose gel electrophoresis was run with the original 8A5 and 9A5 plasmids along with the Miniprep DNA according to protocol (see here). The DNA was also sent off for sequencing by Eurofins.
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. On the second attempt, results from the DNA sequencing confirmed that 8A5 had been transformed into wild type S.oneidensis. This gave us a cell line of Mr1 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 tag.
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