tasks and project ideas

tasks:

1) Get organized: start and maintain a log of protocols/mistakes. I’ve been making a lot of really stupid mistakes recently, twice in a row I screwed up making agar. The first on friday I left unautoclaved bottles over the weekend and they of course got contaminated, the second I mixed the agar at room temperature and since agar isn’t soluble at room temperature the bottles got varying amounts of agar sludge. Luckily the second batch was salvagable by microwaving, mixing, and aliqouting into new sterile bottles. I’m going to switch to a linear lab book (I’ve been using two pads for the last couple, one for the plasmid transformation project, one for the comM project), and need to finish my table of contents for all of my experiments up to this point.

2) Reread grants for project direction/ideas. I need to figure out what I want to do my masters research on

I’m not very interested in the uptake mechanism, partly because it seems specialised to our bacteria. There are a lot of interesting and important aspects to it and there could be significant discoveries, but I don’t think it interests me.  Regulation seems more widely applicable, especially since many of the competence genes only known functions are for competence yet some are widely conserved in other bacteria not known to be competent. No one has good evidence for why as far as I’m aware. The theory I like best is that we can’t detect natural trnasformtion in the lab for most of these bacteria but under certian conditions in the wild competenc eis induced. A group recently found that chitin induces competence in Vibrio cholerae. In a way it’s like the great plate count annomoly, we can only culture a small amount of bacteria, but we know a lot more exist and can grow under some conditions we are not privy too(My roommate in the finlay lab knows a lot about this from working in Allen-Vercoe lab at guelph and has been teaching me a few things)

3) Benchwork: I need to get back to labwork: I made PSU20-pdsred construct last week that glowed when transfered to e.coli but I didn’t recover any Haemophilus mutants. I used much to low a concentration of glycerol so I’ll need to change that next time. I also need to chracterise the transformation frequencies of comMdprArecJ, comMdprAexoI, and comMrecJexoI, although not very important strains I only got numbers from these strains once. I should also do a run of exoI and comMexoI again aswell,  I did them last week and got transformation frequencies that were unusually high for everything

DN experiment 60

KW20: 1.25E-03

comM:  2.28E-04

exoI: 2.81E-03

comMexoI: 9.76E-05

I can’t think of how to overcome the problem of how to test phage recombination in actually competent cells. It’s not a test I necessarily want to do, it’s Rosies project, but the results of such a test are unknown and could be informative.  The research associate explained that sxy-1 can be used to circumvent this issue since sxy-1 mutants have a higher proportion of competent cells

Some background: Sxy-1 is hypercompetent when growing in rich media and has a mutation that disrupts the stem loop structure in the 5′ UTR region. The theory is that purines interact with the wild type stem loop to prevent sxy translation, and due to the stem loop mutation purines can’t inhibit sxy-1 translation. Transformation frequency in MIV is not affected significantly by sxy-1

Sxy-1 has a higher proportion of cells that are competent in rich media. So a test of congression in a sxy-1 mutant should yield a lower number of observed over expected, right? This would give an estimate of how many more competent cells there are. This may have been done but I can’t find the info

I’ve been rereading through my entire lab book, from the start. it’s actually been pretty helpful, a lot of the experiments I did in the begining before I knew as much about the lab and system I can put into more context, and a lot of early experiments I had forgotten about. I did an experiment with azathriprine, a purine analogue which inhibits purine synthesis, that elimanted transformation(more accurately lowered it below LOD). I did the experiment using KW20, presumably the purine analague function inhibited transformation, but a sxy-1 mutant should be immune to this effect. The boring result would be that sxy-1 TF is not affected by azathrioprine due to it’s stem loop mutation.

The alternative approach to testing if competent cells are responsible for the 100 fold increase in phage recombination would be to get rid of competent cells. This might be the more feasible approach since the majority of cells are non competent. Introduce some type of DNA that kills competent cells. Hmm. It’s too bad that arsenic paper was bunk, if arsenic DNA existed and could be taken up by competent cells it might have been a way to elminated them from MIV culture.

I’e also started thinking about how temporal is competence. Maybe that could be a good direction? Transform cells with a marker for 15 minutes, DNAse, somehow destroy the DNAse, add a second marker and measure the frequency of dbl transformants. If competent cells stay competent for a long time the frequency of double mutants under this condition should be more equivalent to double mutants made by transforming with both markers at the same time.  Doing different time points could get a good estimate of time. The methods a bit rough. Just a thought, I really need to figure out what I want to study.

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