Notebook - Week 1

Week
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Monday July 16

To start the week off, Kevin went over some vital guidelines for lab work that we need to follow in order for us to keep organized and avoid wasting time/reagents. Then, since there were just the five of us, we all went into the lab to work on various things. Beini and Faisal did a FastDigest on 9 miniprep products to see if our ligations worked. They digested for 35 minutes instead of the usual 30, in hopes of better results. According to the gel, all the ligations failed. Andrew and Phillip performed a massive PCR of BioBrick parts we haven’t successfully PCR’d yet. Kevin did some reorganization of labwork, such as analyzing previous gels to check for digestion success.

Tuesday July 17th

This morning, Kevin and Andrew met with Dr. Smith to discuss gene sequences for cohesins and dockerins. Phillip and Andrew continued the gel analysis that Kevin started yesterday. Kevin, Faisal, and Beini worked on making primers for our next order. We planned to put in a rush order for only the primers that we would need to make a working GFP construct. In order for the primers to arrive tomorrow, we had to get our order in by 3 pm, so when everyone came back from lunch at 2 pm, we put all our heads together to grind out those primers. However, at 2:52 pm we ran into some unforeseen issues that required some consideration, so then we decided to chill out and just put an order in for 3 pm tomorrow. In the lab, we rehydrated a RBS BioBrick part and transformed it, liquid cultured some BL21 and XL1 Blue, and added a promoter to fliC1 using PCR.

Wednesday July 18th

Today, we put in our rush order for primers so that they hopefully arrive tomorrow. Kevin and Maggie met with a representative from KEDCO (Kingston Economic Development Corporation) to ask for advice on how to garner support for SynthetiQ from the local community (eg. educational institutions, dancers/choreographers). Later in the day, they also met with Dr. Allingham to discuss molecular modelling with a software called Coot, that we might use for modelling our chimeric flagellin. As for labwork, we made liquid cultures of the RBS from yesterday, made glycerol stocks of XL1 Blue and BL21, and digested fliC1 with SpeI to confirm the presence of that cut site. We also attempted a PCR overlap extension of pfliC1 with three different inserts (GFP, smtA, golB).

Thursday July 19th

Our rush order for primers was supposed to arrive by 5 pm, so we kept our eyes peeled for that the entire day, but alas they did not arrive by that time. For labwork, we re-ran yesterday’s PCR overlap extension and added more dNTPs and polymerase, because from the gel it looked like a lot of primers didn’t bind in the original PCR. We also tried PCR overlap extension using pfliC1 and X/S digests of fliC2 and GFP, with no primers. We attempted our first motility assay by stabbing a colony of DH5-alpha onto a soft agar plate. In the afternoon, we worked on primers for conversion of parts to BB-2 standard, and for parts we didn’t have primers for previously. Today was also Kevin’s birthday! Happy birthday Kevin.

Friday July 20th

For our motility assay of DH5-alpha, we got a nicely shaped circle ~1.1 cm. However, DH5-alpha is not supposed to express native flagella, so we will have to look further into this. For our next motility assay of TOP10, we decided to try one soft agar plate and one LB plate. We also divided the plates into quarters and tried four different levels of stabbing (i.e. from shallow to deep). For overlap PCR extension, we ran another round of PCR for pfliC1 + fliC2 + GFP, this time just adding a bit more polymerase and not dNTPs (because they probably didn’t get used up). Then we ran a gel of all the PCR overlap extensions we have done so far. In the afternoon, Kevin showed us how to do a gel extraction, and cut out bands of fliC1 + GFP and fliC1 + GolB from the aforementioned gel. For the rest of the day, Phillip did some filming for our project overview video as we took turns speaking for the camcorder.