Team:BYU Provo/Notebook/CholeraDetection/Summerexp/Period3/Dailylog


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Cholera Detection July-August Notebook: July 29 - August 4 Daily Log

Cholera Detection


By sad, frustrating experience, we’ve surmised that there is a problem with the patches of TT9901 and TT9907 to which we keep returning for experiments. We received these strains from the John Roth lab at UC Davis as three small glass stabs. Whenever we plate them on LB directly from the stabs, we always get spotty growth. We believe that we’ve been ignorantly selecting for host cells that are not susceptible to bacteriophage lambda. To correct this, we’ve selected 32 colonies from 3 patches: 15 colonies from one patch of TT9901, 15 colonies from one patch of 9907, and 2 colonies from our control strain that lacks the lambda lysogen, TT25281. We’ve started these as overnights. Tomorrow, we’ll freeze down one mL of each strain, and with the remaining culture, we’ll make top agar lawns using X1 top agar. In the middle of these lawns we’ll add a drop of hydrogen peroxide, which is known to stress cells enough to cause lambda to convert its lytic cycle. Any lawns that show plaques relative to a non-H202 control top agar lawn will be strains that we know have lambda and can become induced to lysis.


It’s a good thing we chose to run this experiment with 15 colonies from each strain, because very few showed plaques. Specifically, the 9th, 10th, and 20th strains had plaques on their lawns and no plaques on the non-H2O2 control. Numbers 9 and 10 are bacterial progeny of TT9901, and number 20 descends from TT9907. Since we’ve frozen these down, we now can return to source strains that reliably contain bacteriophage lambda that is inducible to lysis!


Submitted sequencing for Qrr4/RFP clones. 2 clones had 1 nucleotide difference in the Qrr4 promoter section. We concluded this was a negligible difference. One of these were frozen down as pIG89.