Team:British Columbia/Notebook/Week 11

beta-Pinene

Still working on getting the original synthase onto a yeast plasmid containing GPD promoter, she re-dos the ligations using a 6:1 ratio instead of 3:1. She then transforms the product into E.coli. Vicki also digests and ligates the original synthase and the yeast plasmid (GAL) together and transforms that into E.coli.

1,8-cineole

There are colonies on the yeast plates! They remained very small even on the fifth day after plating, but the fact that they were present means that Jacob can proceed with the gc-ms.

Only the synthases that had undergone SDM were used to transform the yeast so far. In order to check that there was no change in the expression due to the silent mutations that Jacob introduced with the SDM, he prepared shuttle vectors to get the original synthase into yeast.

The pgxe-cineole biobrick seems to be correct, so Jacob plans to make a glycerol stock of it, as well as submit the biobrick to the parts registry. Hurrah! We have a part!

The pg-cineole biobrick has been unsuccessful up to this point, but another round of PCR to attach the biobrick suffix and prefix was quite successful. Previously, Jacob was using the annealing temperature of just where the primer would bind to the original synthase, but it was pointed out that in a PCR reaction, all subsequent same-direction amplifications after the first one attach fully, raising the Tm. Using the new Tm, the PCR worked far better. The ligation and transformation were also successes.

Characterization: Limonene Synthase (LIMS1) - Part BBa_I742110

Vicki digested and ligated following samples:

1. limonene synthase + yeast plasmid with GPD promoter
2. limonene synthase + yeast plasmid with GAL promoter

She then transformed the products into E.coli cells. Waiting for results...

Characterization: GPD-GFP

We looked at the expression of GFP as regulated by the yeast GPD promoter under a fluorescence microscope...