Blog

Sorry there haven’t been updates in a while. Things have been a little difficult in the lab recently. I’m working on a powerpoint presentation for Rosie, I might post some slides on the blog after I speak with her.

I mostly just want to jot down a thought I had during the last lab meeting. When I started in the lab I worked on dprA and comM, two competence genes that have significant negative effects on transformation when knocked out. We weren’t sure what the roles of these genes were. dprA for instance might protect single stranded DNA from degradation by exonucleases or it’s role might to bring uptaken DNA to recA. Because dprA is very widespread and highly conserved in bacteria the answer would relatively important. We used a double mutant approach to explore, but this approach has some limitations.

One of the difficulties in investigating the roles of these two genes is that we do not have good tools for investigating what goes on in the cytosol, mostly because it’s difficult to distinguish the uptaken DNA from chromosomal DNA. Transforming cells with a specially designed PCR product that has little homology to chromosomal and critically includes several primer sites that do not have homology to the chromosome. In a recA- background no recombination is possible so all the DNA that’s has been taken up and translocated in the cytoplasm will persist in the cytoplasm, being degraded by exonucleases. We have an effective protocol to rupture the outer membrane(useful for studying the periplasm),  adding DNAase after this step should degrade all the DNA that is not in the cytosol. Afterwards a DNA prep(perhaps with a wash to get rid of the DNAse) can recover just the DNA in the cytosol which will be mostly chromsomal

If dprA and comM roles are to protect DNA from degradation, the PCR fragments fed to the cells should on average be shorter in knockouts. By using primers unique to the ends of the PCR fragment, qPCR would allow for quantification of how many intact strands remain. Having a stepwise system of primer sites on the PCR fragment would allow investigation of questions such as how much degradation is going on and where.

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