Team:British Columbia/Notebook/Week 8

July 24 2011

(-)-limonene

• Results from transformation:
  • Positive control: TMTC
  • Negative control: no colonies
  • pADM743: TMTC

• Set up O/N culture for mini-prepping
  • 4 tubes: 3 colonies picked + 1 control

Track: Beta-pinene synthase

Marianne gel verified her cPCR using both G1004/G1005 and VF2/VR primers, but they showed no bands.

July 25 2011

On July 25th, Gurpal performed a yeast transformation. He transformed his purified pBS and pKS into 2 separate wildtype yeasts. He followed a protocol, left the cells to incubate at 30 degrees Celsius at 3:10pm, heat shocked them for 20 minutes at 42 degrees Celsius and then plated them at 5:30pm. The plates were marked with URA so that the plasmids could grow on them. Then, they were incubated in a 30 degree Celsius incubator.

(-)-limonene

• Miniprepped pADM743

Track: Beta-pinene synthase

Marianne's transformations (yeast plasmids + synthase) show a few colonies each on 3 plates (original-his-GAL, SDM-no-his-GPD, SDM-no-his-GAL. Marianne conducted a colony PCR of these yeast plasmid and synthase transformants and gel verified them. The gel image shows bands at the correct lengths (~500bp), indicating that the ligations were successful! We have beta-pinene on yeast plasmids!

July 26 2011

Track: Beta-pinene synthase

Marianne started on the Biobrick assembly; she restriction digested and ligated synthase and pSB1C3 backbone. Marianne set up an O/N culture for the yeast plasmids.

Marianne and Jacob are putting their parts in the biobricks plasmids!

Alpha- Pinene

July 27 2011

On July 27th, Alina observed the cells and noted that the transformation was a success.

Track: Beta-pinene synthase

Marianne transformed the Biobrick ligations and miniprepped the yeast plasmids. Marianne also transformed the miniprepped yeast plasmids(SND, SNL) into wildtype yeast, pKS yeast, pBS yeast.

1,8-cineole

• The previous colonies of E. coli that should have had the 1,8-cineole gene on pSB1C3 were shown to have different sized plasmids after overnight cultures and minipreps. Of the 5 colonies, 4 had plasmids a little over 2 kb in size, and one had a plasmid around 5 kb in size. The 1,8-cineole gene is 1.7 kb and the pSB1C3 plasmid that is left after the restriction digest is 2 kb, so the product should have been around 3.7 kb. A restriction digest with ECORI and PSTI showed only one band for the 5 kb product, and the 2 kb product showed bands at about 1.7 kb and 700 bp. All of these were grown on chlor plates and chlor LB broth for the overnight culture. A gel of just the pSB1C3 showed a product well below the 500 bp mark on 1 kb ladder.

• This was rather disconcerting, so Jacob decided to run another biobrick PCR and try the ligation again.

July 28 2011

beta-pinene

• Biobrick ligations

July 29 2011

beta-pinene

• Biobrick transformation

July 30 2011

Track: Alpha-pinene synthase

Joe finally received his primers for PCR amplification of his SDM-alpha pinene synthase necessary for the ligation into yeast and biobrick plasmids. He did a PCR using these primers. However, no PCR products were seen after gel verification. The annealing temperature may have been too low at 55 degrees Celsius. Therefore, he needs to increase the annealing temperature to 60-72 degrees Celsius, the optimal temperature for Phusion Hot Start Polymerase.