Team:British Columbia/Notebook/Week 7

July 18 2011

Track: Beta-pinene synthase

Marianne's sequencing failed for all of P2SDM1, P2SDM2, P2SDM3. Marianne, very frustrated at this point, decided that she doesn't want to waste any more time on sequencing. So she restriction digested the miniprepped plasmids to see if the plasmids get cut (if the site is still there, then the vector should cut and give a single band).Gel verification of the digests suggested that the site has been removed (show bands that are identical to the uncut template/original vector).

July 19 2011

1,8-cineole

• Despite not getting any sequencing results, the restriction digest gels clearly showed that there were no cut sites in the synthase, so Jacob decided to try getting the synthases on biobricks. Today, Jacob ran a biobrick PCR to add the proper prefix and suffix to his synthase, but it did not work.

Miscellanea

• New ampicillin plates were made, and the pag 413 GPD plasmid (for putting our synthases in yeast) was amplified. After the plasmid was amplified, the team decided to use another plasmid, making the amplified pag 413 GPD plasmid irrelevant for the project.

ERG20

Gurpal was working with the erg20 gene and trying to synthesize the erg20-2 mutant from it on July 19th. After ordering and receiving primers (forward and backward), his goal today was to isolate the erg20 gene from wildtype yeast. Three tubes were prepared for the PCR, each containing a different amount of yeast genome, and the PRC was initiated at 2:20pm. When the PRC was finished, the tubes were incubated in a 37 degree incubator for 1.5 hours. Afterwards, Jacob ran a gel electrophoresis experiment to determine if the site was cleaved. However, the gel showed that nothing except our ladder. Therefore, this experiment failed.

Fortunately, the erg20 and erg20-2 genes arrived from France on pNEV-N plasmids on July 20th! This greatly increased the entire team's morale 10 fold!

On July 21st, Gurpal transformed the erg20 and erg20-2 genes into e-coli to make more copies. The erg20 gene was on a pBS plasmid and the erg20-2 was on the pKS plasmid. He followed the protocol in the lab and made 4 plates: plate 1: pBS plasmid + ampicillin + LB agar plate 2: pKS plasmid + ampicillin + LB agar plate 3: ampicillin + LB agar plate 4: LB agar The plates were grown overnight. On July 22nd, the first plate and second plates were observed to have ln (too many colonies) and the controls were empty - as expected. However, this experiment was not sufficient because there was no positive control.

Gurpal repeated the transformation protocol and used a control with ampicillin resistance. This time, he made brand new ampicillin LB agar plates and plated 4 of them. The plates contained: plate 1: pKS plasmid + ampicillin + LB agar plate 2: pBS plasmid + ampicillin + LB agar plate 3: pg plasmid (we already know it grows in ampicillin) + ampicillin + LB agar plate 4: competent cells + LB agar We expected plates 1-3 to grow single colonies and plate 4 to contain nothing. Gurpal grew the plates overnight for 18 hours. On July 23rd, the experiment was proven to be a success because plates 1-3 contained single coloneis and plate 4 contained nothing.

In the afternoon of July 23rd, Gurpal prepared 5 overnight cultures. tube 1: pKS plasmid + LB broth + ampicillin tube 2: pKS plasmid + LB broth + ampicillin tube 3: pBS plasmid + LB broth + ampicillin tube 4: LB broth (control) --> he expected it to grow nothing tube 5: LB broth + ampicillin (control) --> he expected it to grow nothing On July 24th, the experiment was proven to be a success because the control tubes (4 and 5) were clear liquids. This meant that nothing grew in them.

In the afternoon of July 24th, Gurpal mini prepped his overnight cultures using a PureLink Quick Plasmid Miniprep Kit and isolated pBS and pKS DNA plasmids. In particular, the concentration was determined using a nanodrop: pBS: 100.5 microg/microL pKS: 90.0 microg/microL pKS: 69.2 microg/microL In order to verify the transformation was a success, Gurpal and Jacob performed a gel electrophoresis experiment. The gel columns were: column 1: purified pBS plasmid column 3: purified pKS plasmid column 5: purified pKS plasmid column 7: 1kb DNA ladder The results were columns 1, 3 and 5 each showed up around 7.5 kb. This proves the transformation is a success because both the pKS and pBS plasmids were approximately 7 kb in size.

3-Carene

Daisy has been trying to put the his tagged truncated 3-carene synthase into the yeast plasmid, PA415GPD. This has been more difficult than anticipated. The primers used to PCR out the truncated 3-carene synthase seem to produce a smear-like band at around 1.7 kb. This is the correct size range for the truncated synthase but it is not a clear definite line. Daisy may need to adjust the annealing temperature of her primers to try and get a specific band. At this point, she is going to try and digest and ligate this non specific band into the yeast plasmid. She will try purification methods of gel extraction and PCR purification to try and make the ligation work.

Daisy is also starting on the characterization of a limonene synthase in bacterial expression systems.

July 20 2011

Track: Beta-pinene synthase

Marianne PCR'ed off the original and the SDM'ed synthase using yeast primers (contains XhoI and SpeI restriction enzyme sites) in order to put it in the yeast plasmid. Marianne also PCR'ed off the SDM'ed synthase using biobrick primers (contains EcoRI, XbaI, SpeI, PstI restriction enzyme sites) in order to put it in the psb1C3 backbone. Gel verification for both PCRs shows bands at the correct length!

(-)-limonene

• PCR off the synthase using yeast primers (contains SpeI and NotI restriction enzyme sites) in order to put into the yeast plasmid
• Gel veriification shows 4 bands - PCR failed

• SDM PCR the synthase using new primers

July 21 2011

(-)-limonene

• DpnI digest yesterday's SDM PCR products at 37C for 4 hours
• Gel verification of SDM PCR products show no bands. PCR failed.

• PCR off the synthase to put onto yeast plasmid
  • Troubleshooting: Use gradient, from 64C to 72C
• Gel verification of PCR products for all samples show 4 bands (same as yesterday). This may indicate, again, unspecific binding.

Track: Beta-pinene synthase

Marianne started to assemble the biobrick part. She digested the linearized psb1C3 and the synthase (part) with EcoRI and PstI using Biobrick Restriction Digest protocol. She then ligated the backbone and the part using Biobrick Ligation protocol. Finally, the ligation was transformed into DH5 alpha competent cells.

1,8-cineole

• Assembling biobrick part.
  • Second PCR did not work
    • Third PCR set to run overnight

July 22 2011

Human Practices Laura is very excited to meet with Mrs. Anderson on Monday to discuss our teams involvement in the Future Science Leaders program at Science World! Science World British Columbia is a non-profit organization which engages British Columbians in science and inspires future science and technology leadership throughout our province. The Future Science Leaders is a program offered to high school students who are keen and bright and want to further their understanding of science. Through weekly meetings the program will cover several difference sciences such as biology, mathematics, technology, physical sciences, earth and space, and technology. We as an iGEM team have the privilege of presenting our project to this group of high school students and their parents. We will also be educating the students on the diverse applications of synthetic biology and give a brief tutorial about what synthetic biology is! There is also an opportunity to promote the idea of a high school iGEM team for next year. I am anticipating this meeting and hope to get some feedback and advice for other related Human Practice projects.

(-)-limonene

• Re-do PCR to get the synthase off of the pET101 plasmid onto the yeast plasmid
  • Troubleshooting: Decrease the extension time to 30sec (15sec/1kb) with an annealing temperature of 70C

• Transform original plasmid (pADM743) into DH5alpha cells

Track: Beta-pinene synthase

Marianne conducted colony PCR on the colonies from biobrick plates (using G1004,G1005 primers), but gel verification showed no bands. Therefore, she cPCR the same colonies from biobrick plates using a new batch of G1004/G1005 primers as well as VF2/VR.

Marianne also restriction digested the yeast plasmids and synthase and ligated the following 8 versions together:

• original + his-tagged + GAL
• original + his-tagged + GDP
• original + no-his + GAL
• original + no-his + GDP
• SDM + his-tagged + GAL
• SDM + his-tagged + GDP
• SDM + no-his + GAL
• SDM + no-his + GDP

Marianne then transformed the ligated samples. Marianne had a very long day...