July

Week 1 (July 4 – 10)

We prepared stock solutions of MgCl₂, CaCl₂ 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 E. coli 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.

Week 2 (July 11 – 17)

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.

Week 3 (July 18 – 24)

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.

Week 4 (July 25 – 31)

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 E. coli 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.

August

Week 5 (August 1 – 7)

We miniprepped the level 1 PET biodegradation assemblies out of TOP10 and transformed SHuffle with them. We performed a swell test of both hydrogels, and observed that the CMC hydrogel flaked easily, while the CMC-CA hydrogel became a syrup-like gel and absorbed the water very well. Our bioremediation parts finally arrived, so we began domesticating them. We also did a level 1 JUMP assembly of the untagged sfGFP control.

Week 6 (August 8 – 14)

We did domestications of our new tags; the His-, SUMO-, CBD-, and SB7 Si-tags. Finally, our GMRA was approved, so we could transform our biosensor assemblies for expressing transcription factors. We then prepared lysates of our cells, and ran them on an SDS-PAGE. We performed colony PCRs for all our PET biodegradation related parts, and most came out as a success. We did another assembly of all the failed assemblies and transformed them into TOP10.

Week 7 (August 15 – 21)

We performed all Bioremediation and Directed Evolution related level 1 assemblies, but they all failed, so we began troubleshooting. We spent all week troubleshooting this, and found out a batch of our competent BL21(DE3) cells was at fault. We also domesticated SELIS parts for directed evolution. We began several trials of in vitro transcription. We observed an anomalous result which exhibited a color change, but no fluorescence.

Week 8 (August 22 – 28)

While troubleshooting the biosensors, we found out we had used 10,000x more lysozyme than needed while making the lysates, so we began making them again with the correct amount. We retransformed cells with the Bioremediation level 1 assemblies, and got some white colonies. We did colony PCR of these colonies, which yielded lanes with no bands. We tried this again and got the same result, and found that none of them had the insert we were looking for. We began troubleshooting the assemblies and transformations again.

September

Week 9 (August 29 – September 4)

We realized there was an issue with the strain we were using to express the transcription factors for biosensing, as we were trying to use TOP10 for protein expression. We miniprepped the constructs out of TOP10, and transformed into BL21(DE3). We also did error-prone PCR of our transcription factors and metallothioneins for directed evolution. We also retried a few level 1 assemblies and transformations under different conditions, and they were a success. We decided to break down the remaining level 1 assemblies into four batches, to prevent overusing consumables on batches that would fail, and did the first of four batches. We also did the first PETase immobilisations and began testing PETase activity.

Week 10 (September 5 – 11)

This week, we needed to move labs, and the new lab had more restrictions on the number of people we could have in. We prepared lysate of the transcription factor expression cells, made new aliquots of competent BL21(DE3) cells, did PETase induction from SHuffle liquid culture and did the second and third batches of level 1 assemblies. We also prepared plates with AgNO₃ for directed evolution, and made level 1 assemblies of all error prone metallothioneins except P. fluorescens and C. sapidus. We removed the stop codon from MHETase using PCR. We transformed the first batch into BL21(DE3) cells, sonicated them, and checked for batch 1 protein expression with SDS-PAGE. We continued trying different variations of biosensor trials, and ordered in new constructs expressing different aptamers.

Week 11 (September 12 – 18)

We did more sonications of cells expressing PETases, and finished the activity assays. We did HPLC for fast PETases, redid the third batch of assemblies and did the fourth batch and transformed them into TOP10, did colony PCR of the second batch. We transformed the second batch into BL21(DE3) cells, sonicated them, and checked for protein expression with SDS-PAGE. We tried more biosensor trials with the new constructs, but we could still not get any fluorescence results. We plated the error-prone PCR metallothionein expression constructs on the AgNO₃ plates.

Week 12 (September 19 – 25)

We had organized the batches into order of priority, and as we had dropped a few experiments, there were quite a few assemblies in the third and fourth batches we didn’t need anymore. For those we did need, we transformed them into BL21(DE3) cells, produced lysate and checked for protein expression. We immobilized our CBD-tagged proteins on the cellulose hydrogels and baked them to bind them. We did HPLC of MHETase. We continued troubleshooting the biosensors.

October

Week 13 (September 26 – October 2)

We performed the N- and C-filler characterization experiment. We prepared and submitted hydrogel samples mixed with solutions of heavy metals for native-MS and ICP-MS.