O/N transformations a success

On the thirteenth I came into the lab, grabbed some strains out of the -80 and innoculated 3ml of sBHI, immediatly transformed with 3ul of x-somal DNA I had preped a few weeks earlier, and threw the tubes into the 37 degree roller. Yesterday all the cultures had grown nicely so I poured and innoculated the appropriate antibiotic plates, and put them in 37 again. I just checked the plates today and everything looks good. 0 colonies grew on the no DNA control plates.

Here are the strains:

ComM:tet(RR 1191)  + DprA:kan DNA(RR600)

ComM:tet (RR1191) + DprA:spec DNA(RR3118)

ComM:spec (RR 3121) + DprA:kan DNA(RR600)

ExoI:tetRecJ:kan (RR3025) + ComM:spec DNA(RR3121)

I’ve made the ComM-DprA- double mutant in several different combinations, but that was mostly  a contingency plan incase ComM:specDprA:kan didn’t work. I want to use that strain for testing because all my other ComM mutants use the spec-r cassette, and the DprA mutants KO’s use kan, so it won’t introduce anouther variable. If I feel up to it I’ll test the different double mutants against each other to see if the different antibiotic markers make a difference on transformation frequency, but that’s not a priority.

I’m pretty sure I already have a DprA:kanComM:tet strain but I haven’t been able to verify it by PCR due to a lack of ComM:tet primers. I have a single transformation freq for it, so if the new strain has the same freq it’ll be more circumstantial evidence that it’s real. I did a quick little test in this experiment to pile on the circumstance, I know the suspected DprA:kanComM:tet strain grows on T/K plates, so I transformed ComM:spec with DprA:kanComM:tet DNA and plated on T/K/S. No colonies should ever grow if it’s what I think it is, and none did.

I’ll restreak single colonies on plates today and do colony PCR on them tommorow. I’ve been having issues with spec, and from talking with Josh and doing some reading online the problem could be specs mechanism of action. It binds to ribosomal protein, and a point mutation could disrupt this binding. In E. coli the mutational rate of resistance can be as high as 10-6. Since the transformation freq of these mutants is low already, I’ll check 3-4 different colonies just so I don’t pick badly and get unlucky. The fact that no colonies grew on the no DNA control is a good sign but I’ve been burned before so might as well be safe. Maybe instead of doing No DNA controls I should use DNA that doesn’t have markers on it as a control, incase the addition of DNA does something to the cells.

ref: DN exp #21


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