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From 2011.igem.org

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.

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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.

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