Team:British Columbia/Notebook/Week 4

June 27 2011

We created a set of lab rules due to common mistakes made in the lab over the past few weeks.

Vicki and Marianne will having training sessions throughout the week to show team members without much wetlab experience how to PCR, cast and run a gel, digest, ligate, transform, design primers, send miniprepped products to be sequenced.

Competent cells - tested, and they're competent! Good job Gurpal and Sam!

Modeling: Jacob obtained the ArcGIS program. Gurpal wants to create a wetlab flow chart to match the modeling.

Sent DspB (part from last year) to Grinnell College (a new iGEM team this year who requested our part) in the United States.

alpha-pinene synthase

• SDM-PCR worked! (Used the QuikChange SDM Kit protocol).

3-carene synthase Daisy needs to make custom primers for sequencing purposes because the mutagen site is in the middle of the coding sequence and her flanking primers will only sequence 700-800bp into the sequence.

beta-pinene synthase

• Resuspended SDM forward and reverse primers

(-)-limonene synthase

• Resuspended SDM forward and reverse primers

1,8-Cineole Ran successful SDM-PCR to remove a restriction site.

June 28 2011

Training: went over PCR protocol (SDM specific)

beta-pinene & (-)-limonene synthase Ran SDM-PCR

3-Carene Daisy did a single digest with EcoRI-HF to test if the EcoRI site is in the synthase or not. Chris Keeling emailed Daisy the sequence he used for the truncation of the 3-Carene synthase. Daisy ordered primers to do the sequencing.

June 29 2011

Training: The plan was to make kanamycin plates, but this was at a stand-still as we had troubles locating the tube of kanamycin sulfate salt.

beta-pinene & (-)-limonene synthase

• Verified yesterday's SDM-PCR products through gel electrophoresis. No bands were present in any of the lanes.
• Troubleshooting: The dNTP could have been too old/contaminated (we had troubles with this particular tube of dNTP from last year); the concentrations for the reagents used were not optimized - Marianne researched protocols and optimized the SDM-PCR protocol. We (Vicki and Marianne) will test it tomorrow.

1,8-Cineole Jacob transformed competent cells with his SDM-PCR product. Also ran SDM-PCR on variant of original synthase.

June 30 2011

beta-pinene and (-)-limonene synthase

• SDM beta-pinene optimized protocols online (template amount, primer amount, amount of MgCl₂, and amount of DMSO).
• Gel verification shows no bands.

July 02 2011

1,8-Cineole

Jacob ran Raf's miniprep protocol on transformed cells to isolate a plasmid containing the pg-TPS-cin gene, which should be missing a restriction site.

In a group, Vicki, Marianne, Gurpal and Jacob attempted to do a site-directed mutagenesis on June 28th. Gurpal chose to do his on the PgxeTPS-Cin synthase gene. The goal was to remove an internal cut-site. He prepared 2 samples and a water control. ThermoPol was used as a buffer and PFU were used. After adding the forward and reverse primers (and other constituents), we each ended up with ~ 25 microL samples in PCR tubes. We set our samples to run in a thermocycler for about 4 hours with specified conditions. When the cycling was completed, DPN1 was added to each PCR tube and then the tubes were incubated overnight at 37 degrees Celsius.

In order to test our site-directed mutagenesis experiment, we prepared gels and preformed a gel electrophoresis. We did this one hour after the PCR tubes were incubating at 37 degrees Celsius. Unfortunately, our gel showed that our site directed mutagenesis did not work. But, each of us learned what not to do for next time which is always good.