Team:British Columbia/Notebook/Week 15

Jacob, Joe and Daisy purifying the alpha and beta-pinene synthases under Rafael and Alina's supervision.

Protein Purification of Alpha and Beta-Pinene Synthases

Joe and Jacob engaging in a tug of war to get the centrifuge container out of the holder!

What happened to Alina's bench!

Joe expressed the proteins and spun down the cell pellets on the weekend. Now, Daisy and Joe are going to be doing the protein purification, in vitro assay and the GCMS sample preparation for alpha-pinene and beta-pinene. Daisy and Joe took 1-2 grams of cell pellet and sonicated the cell pellet with cell lysis buffer. The samples were spun down and the supernatant was collected. The supernatant was put through a gravity nickel column. It was washed several times and then eluted with His-column elution buffer. The enzymes were centrifuged onto a membrane and then re-eluted with the correct enzyme assay buffer, to allow the enzymatic activity to occur. The enzymes were incubated with geranyl diphosphate (GPP) and the samples were prepared.

Originally, the rate of the gravity filtration of the nickel column was very slow and would mean that we were going to be there for a very long time. According to Derek (alina's lab mate), we would be in the lab until 11:30 pm if we were to do the purification step. We did stay in the lab until 12 am. (Joe, Daisy, Alina, Jacob, Rafael, Marianne went for a team social dinner!)

SDS PAGE of protein expression

Daisy and Joe have been trying to run SDS PAGE of their protein samples to confirm protein expression. However, it looks like the protein purified is too dilute to result in a noticeable band on the 10% gel... We are going to re-attempt the purification procedure.

GAL and GDP Promoters

Biobricks of GAL and GDP promoters were sent to UBC NAPS sequencing. They came back as great sequences. Please see our part and data page for more details.