Team:NRP-UEA-Norwich/Triparental
RESULTS
To transform the plasmids with C- (8A5) and N- (9A5) terminally tagged iron-iron hydrogenase genes into wild type Shewanella oneidensis MR-1 (WT), S. oneidensis with both hydrogenase genes knocked out (Double knockout) and S. oneidensis with the FeFe hydrogenase knocked out (FeFe knockout). The pBAD vector was used as previous experiments attempting to transform Biobricks into Shewanella failed.
We used triparental conjugation firstly, as S. oneidensis MR-1 are not amenable to heat shock. The 2:1 protocol was followed; see here. We used strains of E. 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 2:10 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 protocol 1:C; 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 at Eurofins.
The triparental conjugations were attempted twice, however no colonies of Shewanella were identified when grown on kanamycin plates for any of the combinations of plasmid and strain types. When the method was trouble shooted, an issue was identified at the final transformation step, as before this step when the cultures were spun down they were red coloured indicating the presence of Shewanella. However, when colonies were inoculated from the final plates and spun down these were all white and so were identified as contaminants. Electroporation was performed as an alternative method to try to get both plasmids into the three different S. oneidensis strains. On the second attempt, all strains (except the FeFe knockout with 8A5) had been transformed and could be seen as red colonies on the plate. The agarose gel electrophoresis confirmed that the DNA was of the correct size, but the bands were slightly blurry. Results from the DNA sequencing confirmed that only one of the transformations had worked (8A5 into wild type Shewanella).
Figure 1: Plates showing Wild Type Shewanella oneidensis MR-1 with the C- terminally tagged FeFe hydrogenase genes (8A5) containing plasmid.
Sponsors
