Team:Austin UTexas/Results/Conjugations

• HOME
• TEAM ▼
  • Meet the Team!
  • Collaborations
• PROJECT ▼
  • Description
  • Results
  • Kit Contents
  • InterLab
  • Demonstrate
  • Improve
  • Attributions
• PARTS
• SAFETY
• HUMAN PRACTICES▼
  • Silver HP
  • Integrated and Gold

Conjugations

Close ×

Menu

Part Plasmids Assembly Plasmids Electroporations Conjugations

☰

Navigate Results

Conjugating into S. marcescens

When building the plasmid kit in E.coli, a model organism, electroporation was a convenient avenue for transforming plasmids into the host. However, electroporation protocols can vary for different bacteria species and many may have no protocol developed for them at all. Rather than assuming the burden of developing one, researchers rely on conjugation. This is the transfer of a plasmid from one species of bacteria to the other, and happens in nature. We electroporated the GFP plasmid from our kit into a strain of E.coli that needed extra nutrients from the media it was grown on. Then we grew it along with S. marcescens at different ratios. Samples from the dual cultures were then grown on media without the extra nutrients, selecting for only S. marcescens. The S. marcescens that did grow on the media contained the plasmid, expressing the green fluorescence.

Figure 1: Demonstration via Conjugation into an Serratia marcescens Model. Depicted above are two kanamycin-containing LB plates on which S. marcescens is growing. The top plate contains S. marcescens conjugated with BHR901. The bottom plate contains the controls, in which assemblies known to work were conjugated. The left side shows the plates under normal light; the right side shows the same plates under a high energy blue light.

The results of the conjugation are shown in Figure 1. Although the green coloring of S. marcescens with the BHR kit plasmid (top) under UV light (right) is not as strong as some of the controls, the bacteria is clearly expressing the green phenotype, which it does not naturally have. The figures above contain bacteria that was streaked on media that did not contain additional nutrients, and then was picked and restreaked again to ensure that no nutrients or colonies without plasmids remained. Because the first round of selective plating killed off the samples of only the MFD E.coli that needed the extra nutrients regardless of whether or not they contained a plasmid, they are not included on the plates above. The untransformed S. marcescens and Top 10 E.coli did not have the antibiotic resistance the plasmids coded for and therefore they died on the media because it contained KAN antibiotic. The control plasmid contained a GFP and KAN resistance as well. However, it appears to have allowed the MFD strain to survive on the selective media, as traces can be seen in one of the control plasmid conjugation sections, as well as the sections where MFD plus the control plasmid was plated alone. However, because all the negative controls for the S. marcescens conjugation with a plasmid from the BHR kit died on the selective media, the results are not assumed to be invalidated.