Team:BYU Provo/Notebook/CholeraDetection/Springexp/Period2/Dailylog

7//13

7/12/13

-KK, CH, KP

The big bummer: we could not induce Lambda into its lytic cycle using CRO. On Wednesday we prepared our bacterial sample on agar, with arabinose, and poured the plates. Today the plates showed no plaques. They were all full lawns of bacteria.

Our RFP-qrr4 sewing PCR is going well. A gel showed that on Wednesday we successfully amplified the promoter for Qrr4, and today we started the PCR reaction to sew together RFP and the qrr4 promoter. Our template for RFP came from the 2012 iGEM kit, "Cherry RFP," from Kit Plate 1, well 18-F.

5/15/13

-KP Today we started out by getting photographed. We also watched the phage group’s presentations. It seems as if they have a good start to their project. We didn’t have too much time to do our own experiments, but we did set up an overnight to do mini-prep tomorrow, and we streaked out our plates of lambda phage.

-KK The three phage groups presented today. I realized how very quickly each of our groups has specialized to the point that I have to concentrate deeply to understand what seems to be second nature to members of the phage group. That’s good! It means each of our groups is moving along nicely. The objective of the phage project is to make a library of sizes - the biggest and smallest phage capsids possible using phages that have already been well characterized and approved for medicinal purposes. One group is working on selecting for the largest phage possible, one for the smallest, and another group is developing a method to purify both phages as they are made. Afterwards, we laid out singles of our three E.Coli strains with Lambda prophage incorporated into its genome. Tomorrow Kelton and I are going to go in and check on them after our Intro to Medicine class.

5/17/13

-KK All our singles grew up well, so today we set up 10 PCR reactions + our control to test which, if any, had our vector + CRO insert construct. We also checked the plate that we streaked with Cholera and E.Coli with pIG78 next to one another. We wanted to see if the E.Coli would fluoresce at all in the presence of cholera. Under a UV light we didn't observe any noticeable difference between the streaks of cholera and the streaks of E.Coli.

5/20/13

-KK Clarisse left for Russia on Sunday, so she'll be gone for perhaps a month working at her internship. At the beginning of class we talked about possible community projects. The first project we may want to do is publish a children's book. The other two projects that received votes were: creating a board game, and sponsoring a fun run to raise money for Haiti. I called my friend Redge Ballard, an animation major, to ask if he would be interested in designing the book. We PCR verified our 10 colonies and only colony A had the CRO insert we were looking for. We set up overnights of colony A to do a plasmid prep and then sequence, and we also set up overnights of our three lambda strains to plate tomorrow as a lawn. Once we do that, we hope to be induce lambda to go lytic by plating our E.Coli with CRO.

5/22/13

-KK, KP Yesterday I came in and did a plasmid prep of colonies A and W, which looked promising for having CRO+pIG11 on our PCR readout. Today we confirmed on the spectrophotometer the concentrations of the plasmids. The plasmid from A was present at a concentration of 158 ng/microL, and that of W had a concentration of 62 ng/microL. Yesterday we also plated our three strains of lambda-infected E.Coli, TT9901, TT9907, and TT23281, so today we were ready to trasform the plasmid into these three plasmids using electrophoration. Dr. Grose showed us how to use the electrophorator. We shocked each strain with plasmid from colony A and then a control plasmid separately, for a total of 6 electrophorations. After letting the cells recover for 30 minutes at 37 degrees celsius, we plated about 1 mL of each on LB/Amp plates. Tomorrow, hopefully, we will see a few colonies indicating that they took up our plasmid! Also, we submitted plasmids from A and W for sequencing.

5/24/13

-KK, KP Our electrophoration worked on 2 of the 3 strains - on TT9901, and TT9907, but not on TT23281. Yesterday we went ahead and set up overnights to plate the two successful transformed strains on Amp/Arabinose, but our Amp/Arabinose plates are no longer good, so we need to make more. We redid the electroporation today so that hopefully by next wednesday all three strains should be ready and we should have plates. We'll be testing to see if TT9901, TT9907, and TT23281 with Lambda's CRO gene cloned into them can induce the integrated prophage out of lysogeny and into the lytic cycle.