Difference between revisions of "Team:NRP-UEA-Norwich/Triparental"

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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.  
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To transform the plasmids containing C-terminally strep-tagged & N-terminally strep-tagged <i>hydABC</i> FeFe hydrogenase genes into wild type <i>Shewanella oneidensis</i> MR-1 (WT), <i>S. oneidensis</i> with both hydrogenase genes knocked out (ΔHydABC,HyaABC) and <i>S. oneidensis</i> with the FeFe hydrogenase knocked out (∆HydABC). The pBAD vector was used as the previous experiments attempting to transform BioBricks into <i>S. oneidensis</i> failed (see <a href="https://2016.igem.org/Team:NRP-UEA-Norwich/BioBricks"> BioBrick conjugation </a>).  
 
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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.
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The tri-parental conjugation protocol was initially carried out, as <i>S. oneidensis</i> MR-1 are not susceptible to heat shock. We used strains of <i>Escherichia coli</i> 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 <a href="https://2016.igem.org/Team:NRP-UEA-Norwich/Protocols"> here </a>. 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 <i>hydABC</i> C-terminally tagged and <i>hydABC</i> N-terminally tagged plasmids along with the Miniprep DNA. The DNA was also sent off for Sanger sequencing.
 
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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).
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Triparental conjugation was attempted twice, but each time no colonies of <i>S. oneidensis</i> 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 <i>S. oneidensis strains</i> (Wildtype MR-1, Double hydrogenase knock out ∆<i>HydABC,HyaABC</i> and FeFe hydrogenase knockout ∆<i>hydABC</i>. On the second attempt, results from the DNA sequencing confirmed that <i>hydABC</i> N-terminally SII tagged had been transformed into wild type <i>S.oneidensis</i>. This gave us a cell line of MR-1 containing an arabinose inducible <i>hydABC</i> gene to be used in following proof of concept experiments (from this point denoted MR-1:HydABC)
 
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<img src="https://static.igem.org/mediawiki/2016/d/d6/T--NRP-UEA-Norwich--shewanella_oneidensis.jpg"  class="showFullSizeImage centerMiddle" />
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Figure 1: Plates showing Wild Type Shewanella oneidensis MR-1 with the C- terminally tagged FeFe hydrogenase genes (8A5) containing plasmid.
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<span class="bold">Figure 1: Kanamycin plates showing successfully transformed colonies Wild Type <i>Shewanella oneidensis</i> MR-1 containing the golden gate construct </span>. This construct contains the <i>hydABC</i> gene cluster, with a C-terminal strep II tag.  
 
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<img src="https://static.igem.org/mediawiki/2016/d/d6/T--NRP-UEA-Norwich--shewanella_oneidensis.jpg"  class="showFullSizeImage centerMiddle" />
 
  
  

Latest revision as of 19:00, 19 October 2016

NRP-UEA-NORWICH iGEM

RESULTS

Aim

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 ).

Method

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.

Results

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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