<p>We prepared stock solutions of MgCl<sub>2</sub>, CaCl<sub>2</sub> and glycerol. We produced overnight cultures of J23100, B0034, L2U2H09, Uac level 0 acceptor and LacZ level 1 acceptor, and then miniprepped them. We also prepared a stock of competent TOP10 <i>E. coli</i> cells, and stored them at -80°C. We also attempted to use PCR to add JUMP fusion sites to a merR part, while removing the stop codon. Unfortunately, this did not work.</p>
<p>We prepared stock solutions of MgCl<sub>2</sub>, CaCl<sub>2</sub> and glycerol. We produced overnight cultures of J23100, B0034, L2U2H09, Uac level 0 acceptor and LacZ level 1 acceptor, and then miniprepped them. We also prepared a stock of competent TOP10 <i>E. coli</i> cells, and stored them at -80°C. We also attempted to use PCR to add JUMP fusion sites to a MerR part, while removing the stop codon. Unfortunately, this did not work.</p>
<h3>Week 2 (July 11 – 17)</h3><br>
<p>We domesticated mut_merR, pbrR, and arsR, but were unable to transform them into cells as our GMRA had not been approved yet. In the meantime, we did a bradford calibration curve.</p>
<p>We domesticated mut_MerR, PbrR, and ArsR, but were unable to transform them into cells as our GMRA had not been approved yet. In the meantime, we did a bradford calibration curve.</p>
<h3>Week 3 (July 18 – 24)</h3><br>
<p>We started PCR of silica tags for PET immobilization, which initially failed due to primer dimers, but then succeeded when we slightly raised the temperature. Our iSpinach biosensor constructs had arrived as well, so we started a PCR of them, which failed a few times and eventually succeeded after enough optimization. We also produced a stock solution of carboxymethycellulose (CMC) at 3% w/v. </p>
<h3>Week 4 (July 25 – 31)</h3><br>
<p>Dr. Marcos Valenzuela-Ortega gave us a stock of cells with a merR O part plasmid after our PCR amplifications failed. We produced an overnight culture of these cells, and miniprepped the plasmids out. We prepared a stock of competent SHuffle <i>E. coli</i> cells. We also began level 1 assembly of PET biodegradation parts. We also transformed the level 1 assembly products into TOP10 cells, plated on IPTG, Xgal, and Kanamycin plates. We made CMC hydrogels, as well as CMC/CA (citric acid) hydrogels. We observed that the CMC hydrogel had grid-like non-uniform square shapes, while the CMC-CA hydrogel was a hard uniform thin layer. We were able to use the 2021 Edinburgh iGEM Team’s GMRA for PETase related transformations, but all biosensor related transformations still needed to wait for GMRA approval.</p>
<p>Dr. Marcos Valenzuela-Ortega gave us a stock of cells with a MerR O part plasmid after our PCR amplifications failed. We produced an overnight culture of these cells, and miniprepped the plasmids out. We prepared a stock of competent SHuffle <i>E. coli</i> cells. We also began level 1 assembly of PET biodegradation parts. We also transformed the level 1 assembly products into TOP10 cells, plated on IPTG, Xgal, and Kanamycin plates. We made CMC hydrogels, as well as CMC/CA (citric acid) hydrogels. We observed that the CMC hydrogel had grid-like non-uniform square shapes, while the CMC-CA hydrogel was a hard uniform thin layer. We were able to use the 2021 Edinburgh iGEM Team’s GMRA for PETase related transformations, but all biosensor related transformations still needed to wait for GMRA approval.</p>
