Team:Pitt/Skin Probiotic/Dehydrogenase/Results

From 2014.igem.org

Results for Desaturase

As shown in Figures 1 and 2, the Desaturase Part was successfully isolated, however our ligation attempts were unsuccessful and a subsequent transformation plated on LB+CAM plates never yielded successful colonies.

Possible hypotheses for failed ligation:

Active ligase could potentially interfere with successful transformation.
mRFP1 is not linearized with the digestion.
Ligation reaction was unsuccessful and the inserts/backbones remained linearized.

Conclusions

After adding an additional step of heat deactivation of ligation reaction at 65 ˚C, the transformation was still unsuccessful, therefore ruling out the first hypothesis. The mRFP1 is re-digested with EcoRI and XbaI, and subsequently ligation was attempted again. Yet still the transformation yielded no useful colonies, also if this theory was correct there should still be colonies that are CAM resistance and will yield a liquid cell culture. Unfortunately, I did not have time to test the third hypothesis prior to the part submission deadline. I had planned to run the ligation reaction on a 0.8% agarose gel to confirm this hypothesis. If ligation reaction was unsuccessful, a series of bands characteristics of desaturase part and linearized mRFP1 should be present on the gel. If the ligation reaction was successful, than there would be a circular plasmid around 4,000 base pair long. In the future we hope to successfully clone this system.