Team:BYU Provo/Notebook/CholeraDetection/Summerexp/Period5/Dailylog

Cholera Detection July-August Notebook: August 19 - August 31 Daily Log

8/19/2013

We verified the PCR of RecA and cleaned it up. We were out of pIG12, so we did a plasmid prep of that and a digestion of the two.

After we electroporate a plasmid into TT9907, we do see a few plaques. Perhaps if we perform an electroporation and then assay many colonies, we could find one that has taken up the plasmid AND remains vulnerable to lambda. We performed an electroporation today; in the next few days we’ll grow up many colonies and run a plaque assay.

KK, KP

8/20/13

BIG news, or at least potentially big news: we believe that plating 9907 adjacent to cholera causes bacteriophage lambda to enter the lytic cycle!

I was researching more to do with the RecA SOS pathway in E.Coli when I stumbled across and abstract that communicated evidence that high concentration of AHLs, homoserine lactones, cause lambda to enter the lytic cycle. Since AHLs are the molecules used in quorum sensing, and V.cholerae also releases them, I thought it might be possible that plating 9907 next to cholerae would trigger lambda to go lytic. I streaked a line of TT9907 next to a patch of cholera that I had grown for several days (we’ve noticed that it takes several days to get solid biofilm growth), and then another line away from cholera. The line perpendicular to and running away from cholera grew, while the streak adjacent to cholera did not! See the photo. Now – we need to verify that the cells don’t grew because lambda is killing them, and not because cholera secretes some toxin that does that.

This opens a new avenue of possibilities. We need to discuss things with Dr. Grose, but probably we will abandon the idea of transforming a plasmid into TT9907.

It took about 4 hours, but today I set up the experiment to test whether any of the post electroporation colonies retains the ability to be lysed by lambda. We grew up overnights from 40 colonies! The overnights were ready today, so prepared 40 top agar lawns and added a drop of H202 in the middle to stress lambda to lyse, should lambda still be a dormant lysogen in our host cell. We should see the results of the test tomorrow.

We ran the digested RecA and pIG12 backbone vector on a low-melt gel. Under UV light we cut out a distinct band for RecA. It is ready for ligation. The plasmid, however, was smeared. Clarice is going to re-digest and re-run pIG12.

KK, KP,CH

Cholera was patched next to our E.coli strain that contained the basic quorom sensing genes from cholera (LuxO/U/CqsS) and a reporter gene's promoter Qrr4 reading into RFP. There was no RFP seen to be produced. Very disappointing. We don't know what is wrong with our construct.

Did a 30ul digestion of pIG12 with XhoI and HindIII to have RecA ligated into it.

CH

8/23/13
I can hardly believe it, but not a single plate among the 40 shows plaques! We didn’t get lucky. We really are at a stalemate with this goal to transform a plasmid into these cells. The other option is to transform a plasmid into cells, and later infect them with lambda bacteriophage. It looks like that’s what we’re going to have to do.

We transformed pIG90 (RecA with a pIG12 backbone) into chemically competent cells via heat shock transformation. Dr. Grose thinks it would be best if at this point, the two cholera sub-groups united into one project. We began working with Nathan and Michael to purify biofilm-degrading enzymes. Work has been met with plenty of obstacles for them. Specifically, they are trying to PCR amplify a gene called dispersinB (DspB). They received the template from an iGEM team in France, successfully PCR-ed the gene, and performed a restriction digest. However, something went wrong and running the digested PCR on a low melt, they didn’t see a band. Since then, they had to re-PCR … but the tube with template DNA was misplaced! They’ve had to use a cleaned-up PCR as their template, but they haven’t been able to copy the gene. Michael and I started a reaction for DspB.

KK, KP, CH

8/26/13
The transformation of pIG 90 into DH5alpha was successful, but I let the cells incubate at 37 degrees C far too long – today makes three days. So, I redid the transformation procedure today.

Visualized on a gel, the DspB PCR smeared miserably along the length of its well. Tomorrow we’ll set up the reaction again.

KK, KP

New plans. We have decided to try to manipulate the natural system that E.coli already has for quorom sensing to get a reporter for cholera. Literature suggests that when a quorom sensing receptor protein SidA detects quorom sensing molecules from other bacteria it by some unknown mechanism causes and lysogenic lambda phage to become lytic. We want to use site directed mutagenisis to mutate the recognition pocket in SdiA so that it will only be able to recognize auto-inducers from Vibrio cholerae. The SdiA recognition area is similar to that found on CqsS (cholera QS receptor protein). We hypothesis that if we mutate 3 amino acids to be the same ones found in CqsS, SdiA will become a cholera auto-inducer specific response receptor protein.

CH

8/27/13

We performed PCR of DspB again using a cleaned-up PCR that Whitney did a while back. Hopefully we don’t see the same result as the last PCR reaction.

KK, KP, CH