Team:British Columbia/Notebook/Week 6

Beta-pinene

All 3 transformation plates (P2SDM1, P2SDM2, P2SDM3) have colonies! Marianne set up overnight cultures, mini-prepped them and sent them for sequencing. Hopefully the SDM worked and the beta-pinene synthase gene no longer has illegal cut sites.

(-)-limonene

SDMs have failed thus far. More troubleshooting: use less pfu as the glycerol content in the enzyme could alter the reaction - prepared 3 samples, each with 0.5uL pfu, 1ul pfu, and 2ul pfu, respectively. Also prepare a sample using 0.5 taq polymerase only. Also increased the annealing temperature and elongation time.

Again, gel verification shows no bands. More troubleshooting: use a different cycling condition.

Again, no bands. Something is amiss...

1,8-cineole

After 5 SDMs, transformations and minipreps, Jacob should now have ECORI-less, SPEI-less, XBAI-less, PSTI-less pg-tps-cin and pgxe-tps-cin. The gel of the restriction digest has the correct number of bands. Sequencing is the next step to confirm this result.

IDI1, HMG2 metabolic genes

Yeast colonies were found for HMG2, p330 and Cin, but not IDI1. The yeast transformed with IDI1 did not grow on a URA- plate. This may be because the nutrient marker was mutated in the plasmid, but this would not affect PCR to amplify the gene out. As a precaution, IDI1 was re-amplified.