Team:Lambert GA/Notebook

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Week 1 (Aug 3):
Since we had been having difficulties working with pλR LacI, we discussed the merits of using a different promoter for our tsPurple constructs.

Week 2 (Aug 7):
After the previous week's discussions, we transformed, inoculated, and miniprepped weak pLac (hoping that a weaker promoter would cause less strain on our cells). In addition, we attempted to ligate the tsPurples into 1C3, however the miniprep concentrations had been too low, so we needed to order more of our parts from IDT.

Week 3 (Aug 14):
We came to the decision that pLac would not work when our construct was fully assembled because it is the same promoter that is on the second part of our construct. Due to this, we moved back to pλR LacI and inoculated liquid cultures from a pre-existing plate. In addition, we transformed and then miniprepped our tsPurple constructs, however the concentrations of both pλR LacI and the tsPurples were shockingly low so we decided to run a PCR next week.

Week 4 (Aug 21):
After last week's low miniprep concentrations we ran PCRs of the tsPurples, pλR LacI, and pLac-ClpXP-CI. We also discussed our lab work with Dr. Styczynski and Monica McNerney to determine potential sources of error.

Week 5 (Aug 28):
After last week's PCR we did a cleanup and nanodropped, which yielded good concentrations. We then sent pλR LacI and pLac ClpXP CI for sequencing and ligated and transformed our tsPurple constructs.

Week 6 (Sep 4): Since the previous transformations were unsuccessful (with concerns about contamination and plates with the wrong antibiotic), we decided to re-transform. Since those were successful, we ran a colony PCR, however the gel showed that the colony PCR was unsuccessful. Meanwhile, we transformed our RFP proof of concepts.

Week 7 (Sep 11): We inoculated liquid cultures of pλR LacI and retransformed our tsPurple constructs after the unsuccessful colony PCR, however the transformations were unsuccessful as well so we decided to return to earlier minipreps and start over. After starting over and retransforming, it turned out third time is not the charm since it was unsuccessful again. We decided to go another step back and re-digest from the original hydrated tsPurple constructs. Simultaneously, we had an unsuccessful miniprep of pλR LacI (but a successful one of pLac-ClpXP-CI), so we went back to an old digest and ran all three tsPurple digests and pλR LacI on a gel. While the tsPurples were the expected base pair length, pλR LacI did not show up, potentially because of incredibly low concentration.

Week 8 (Sep 18): After realizing some calculations were off the previous week, we re-inoculated from old transformations. In the mean time, we hydrated amilCP, amajLime, and cjBlue from IDT.

Week 9 (Sep 25): Our county had fall break this week and we were unfortunately unable to access our lab space, which put us back a week in our lab flow. In this time, some of our team members drove to UGA's lab so that we could complete the InterLab study.

Week 10 (Oct 2): We picked up where we left off before fall break and ligated pλR LacI with tsPurple, tsPurpleDAS, and tsPurpleLAA. Unfortunately, when we transformed they were unsuccessful, so we went back and re-digested the separate parts.

Week 11 (Oct 9): In order to troubleshoot our pλR LacI construct, we decided to attempt to recreate the part from the iGEM Distribution Kits rather than our previous stocks. We utilized pλR, an RBS, and LacI that was present within the Kit Plates as listed on the Parts Registry, and navigated through the cloning workflow to build the part.

Week 12 (Oct 16): After running the digests of our finished constructs on a gel, we realized that the pλR LacI was not the correct length. We retried digestion using BSA to stabilize the digestion, but it was still unsuccessful. We had already performed the ligation of pλR LacI during the digest to save time, but safely discarded the ligations after realizing it was unsuccessful. We also prepared and sent R0011 ClpXP CI for sequencing.

Week 13 (Oct 23): We ligated the tsPurple into the backbones for parts submission, using pSB1C3 as the plasmid backbone. The DNA was additionally dried down and shipped. We also transformed purchased plasmids for color expression and miniprepped them. For the construction of the construct, we obtained the promoter from the InterLab Study Negative Control (R0040) but mislabeled it in the lab notebook due to an apparent change in parts list sent to us. We also miniprepped R0011 ClpXP CI again to increase our stock amounts. We digested the promoter and tsPurples for eventual ligation.

Week 14 (Oct 30): We ligated the R0040 promoter to the tsPurples and transformed them into the Keio Strains as well as DH5. We also sent our tsPurples in for sequencing.