Team:Queens Canada/Notebook/Week10


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Notebook - Week 9

Protocol Content

Tuesday July 3rd

To start the day, we examined the gels we ran on Friday to try to decide our next plan of attack for isolating fliC C1. Should we try isolating genomic DNA of BL21 again, perhaps using a different protocol that doesn’t involve spooling the DNA out with a Pasteur pipette? Or should we abandon BL21 altogether and try a different strain, such as HB101? It might be a good idea to set a deadline for isolating fliC C1, and just get the gene synthesized if we are unable to do so by that date. Throughout the day, we continued research in our respective sub-groups, and significant progress was made in the Cohesins and Dockerins group. In the lab, we tried some digestions and ligations of parts with fliC C2, and plated competent cells of BL21 DE3 pLysS, XL1 Blue, and DH5-alpha. After lunch, Kevin brought in mini Canada Day-themed cupcakes for everyone.

Wednesday July 4th

All the ligations from yesterday failed, unfortunately. In the lab this morning, Victor and David tried ligations of fmT and GFP with fliC C2. In the afternoon, Victor and Phillip grew liquid cultures for fliC C2 and the three different strains of E. coli that were plated yesterday. As for research topics, Kevin, Dan, and Andrew hunted for gene sequences for specific cohesins and dockerins. Phillip worked on haloacetate degradation and Beini worked on polymerization of flagellin subunits.

Thursday July 5th

Morning newsflash from Andrew: the incubator was set to 54°C. Our latest digestion-ligations didn’t work... now we don’t know whether it was because of the cranked up incubator or some other reason. For the future, we should either use the 37°C incubator room, or just be more careful about brushing the knob of the incubator. A whole bunch of emails were sent today: contacting sponsors, ordering enzymes, requesting genes/plasmids from investigators, etc. Phillip updated the scroll to top button on our wiki. In the afternoon, we received a package from our sponsor Novus Biologicals, containing cool gear like antibody-shaped floaters in fun colours, highlighter-pens, lanyards, and fridge magnets. Our liquid cultures from yesterday worked, so we miniprepped those (fliC C2 + three strains of E. coli). We also attempted a second genomic DNA isolation, this time using a slightly different protocol that did not involve spooling the DNA out and transferring it to another tube. We did this for three strains of E. coli (XL1 Blue, DH5-alpha, and BL21 DE3 pLysS). Those involved in SynthetiQ met tonight and did some bodystorming for our upcoming PCR video, and well as some brainstorming about logistics. We decided that it would be ideal to have 8 dancers involved to effectively illustrate DNA concepts.

Friday July 6th

The lab was buzzing with activity today as we are in full swing with our digestions/ligations and PCRs. Victor, David, Andrew, and Phillip worked in the lab for most of the day, while Kevin and Beini researched the construction of fliD chimeras and motility assays. After lunch, Phillip brightened everyone’s day with chocolate chip cookies that he baked himself. They disappeared pretty quickly. Beini tried designing primers to amplify the fliD constant sequences, but the primers aren’t very ideal, so if we are planning to make fliD chimeras at all, we may want to consider getting the two constant sequences synthesized.