Team:Queens Canada/Notebook/Week11


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Notebook - Week 1

Protocol Content

Monday July 9th

A lot of lab work was done over the weekend. Big up to Kevin and Maggie because--sound the trumpets--they finally managed to isolate the first constant domain of fliC! Hooray! Saturday’s gel revealed a glimmer of hope in the form of a very faint band hovering around 700 kb for one trial of PCR done with genomic DNA of XL1 Blue. Sunday’s gel sealed the deal-- they performed more trials with XL1 Blue genomic DNA, and distinct bands showed up that matched the length of fliC C1. Now, all we need is a working ligation of this gene to be sure that it is our target. To start things off this morning, Kevin gave us a rundown of the busy week ahead. Victor and Andrew went into the lab to get started on making our gigantic hoard of agar plates that we will be needing for all our future ligations. David also went into the lab to do ligations of fliC C2 into plasmid backbones. Research topics today included xylanase (Phillip), isolation of flagella (Beini), and characterization strategies for heavy metal-binding proteins (David). Phillip fiddled around with the wiki and managed to create a sweet background using a photo of Kingston windmills that Kevin took.

Tuesday July 10th

Today in the lab, we performed lots of ligations and heat shock transformations with different combinations of fmT, SmtA, GFP, fliC C1, and fliC C2. We also disposed of old liquid cultures that have been accumulating, as well as a few bottles of contaminated growth medium. A little too much bleach was poured into our liquid waste so some of us got a little heady. Outside of the lab, Phillip worked on potential BioBrick parts for catalysis, David continued updating his heavy metal inventory, and Kevin worked on SynthetiQ sponsorship. Towards the end of the day, a few of us went outside into the glorious sunshine to take some test shots for our gifs. Phillip and Beini held up a white bristol board background while Andrew tried his best to look beautiful, with Kevin behind the camcorder.

Wednesday July 11th

In the lab today, we went through our box of primers to see which ones haven’t been rehydrated. For those that had miniprep products, we rehydrated them for future PCR. We performed digestions and ligations of fliC C1 to ribosomal binding sites (RBSs) of various strength. Outside of the lab, we worked on SynthetiQ sponsorship, primers to order, and fluoroacetate degradation. During our faculty advisor meeting, we updated the professors on our sponsorship progress, SynthetiQ progress, and potential catalysis enzymes. We also inquired about materials that we would like to use for characterization, such as a Waring blender (for isolation of flagella) and a fluorimeter (for fluorescence characterization).

Thursday July 12th

Today, Andrew managed to get his hands on spectinomycin from another lab, so we could finally inoculate mgfp-5, the only part from our last BioBrick order that contained that antibiotic resistance. We made 14 liquid cultures of yesterday’s ligations, of fliC C1 to strong/medium RBSs, although some of the plates just had a few stray colonies on the edge that probably don’t contain the ligated plasmid. Victor and Andrew made soft agar plates for our motility assays in the future. Outside of the lab, we worked on things like: our next round of primers (Andrew), SynthetiQ paperwork (Kevin), documenting new lab protocols (Victor), wiki design (Phillip), heavy metals (David), and expression of flagella by different E. coli strains (Beini).

Friday July 13th

Out of our 14 ligations that we liquid cultured yesterday, 8 worked, so we miniprepped those. We also tried ligating fliC2 and a terminator into a plasmid containing a RBS + fliC1. Victor, our lab manager, is taking his two-week vacation starting on Monday. David, who is very competent and useful with regards to labwork, will also be away next week. So today, the rest of us spent time becoming knowledgeable about lab things, so that hopefully next week things in the lab can still run smoothly.

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