Team:Queens Canada/Notebook/Week8


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Notebook - Week 8

Protocol Content

Monday June 18

In the morning, we looked at different protocols for colony PCR and from those, designed our own protocols, hoping at least one of them would work to amplify our fliC gene. Andrew, Victor, and Beini went into the lab to do PCR of fliC1, GFP, CFP, and PbrR. In the afternoon, Kevin brought in a delicately moist carrot cake topped with a smooth, rich, cream cheese-based icing. After everyone on the team had their share, we brought it over to the Chin-Sang lab for them to dig in. Aaron, David, Dan, and Kevin went into the lab to do colony PCR (which proved to be quite mind-numbing) and make a super gel of our latest PCR products. The rest of us spent the afternoon working on sponsorship and reading about chimeric restriction endonucleases.

Tuesday June 19

This morning we received our second shipment of BioBrick parts in the form of 31 agar stabs. David, Phillip, and Beini worked on inoculating all 27 of the plates we had left with our parts, while Victor and Andrew worked on making more LB plates. Dan and Kevin did more research on chimeric restriction enzymes. After lunch, we all sat down to put our brains together to evaluate the feasibility of making our own chimeric restriction enzymes. Seeing as our deliberation of BioBrick fusion standards has dragged on long enough, Kevin devised a way to decide which one we are ultimately going to use: everyone picks a standard, formulates a convincing argument for why we should use that standard, and then we show and tell! We only had time for David to present his case on the Silver fusion standard (RFC 23), which he did with no shortage of finesse, but we will continue this exciting debate tomorrow!

Wednesday June 20

This morning, we finished off the fusion standard mini-presentations. The final verdict? Tom Knight’s BB-2 standard (RFC 12) would be the most ideal for our purposes. It would be neat for us to test it out to see if it works, since it hasn’t been widely adopted by the synthetic biology community (yet). In the afternoon, Kevin and Maggie played around with Gromacs, a protein modelling software that Dr. Campbell recommended. In the lab, David, Victor, Faisal, and Beini had an assembly line going to prepare 31 liquid cultures. They also tried colony PCR using a different method lent to them by Jun (a Ph.D. student in the Chin-Sang lab).

Thursday June 21

In the morning, we searched the web for genomic DNA isolation protocols to use for our colony PCR. So far, we have not yet been successful in amplifying our fliC constant domain 1 gene, which is more than a little irksome. In the lab, we tried colony PCR with 5 ul of template DNA rather than our usual 1 ul. Faisal and Phillip spent ~2 hours laboriously labelling 31 Eppendorf tubes and spin columns in preparation for mini-preps galore. After lunch, we compiled a summary document of all the BioBricks we have ordered to date, to keep track of our progress for each part. We also discovered that the newspaper article about us got posted to the Kingston This Week website, featuring an excellent photo of Phillip, Andrew, and Kevin looking very happily down at a plate that spelled out QGEM in RFP bacteria. However, there was a typo with Andrew’s last name, spelled as “Paham” (now he can’t even show the article to his Phamily, har har har).

Friday June 22

Today, we ran a PCR and gel of five parts with similar annealing temperatures: mCerulean, mCitrine, and variations. We lowered the melting temperature and raised the number and length of cycles, hoping to increase the sensitivity of our products. These parameters seemed to work well, although we did get some unwanted products on the gel. While Faisal was making the Master Mix, he noticed a large particle floating around in our bottle of ddH2O, reminiscent of newt poo... time to get a fresh bottle. We made a few errors while we carried out the PCR, but always caught them in time... Friday labwork is the most fun! However, we are running out of Hot Start polymerase, so we can’t afford to make too many silly errors. Kevin had a meeting with Melissa Mahady Wilton from the Kingston School of Dance about our SynthetiQ project. She might be able to help us get an artist-in-residence, which would greatly facilitate the actual creation of our dances.