Friday, May 8, 2009

Finished the "Dilution and Plating Techniques" assay a while ago. We were all given 1mL of E.coli cells to dilute and plate out at our whim. Plate, shake, and incubate. If the results are the same, then the differences in techniques are not significant and our experiments can continue as usual (I assume). If we all get beserko-different numbers, we'll have to do more "Technique" assays. At least I'll get plenty of practise.

I guess I'll explain my project in more detail. Our lab is interested in transformation of bacteria. If you took Biol 112, as I did, you'll remember this (or you should). Certain bacteria can take up free-floating DNA from their environment and make it a part of their own chromosomal DNA (the new DNA has to be very similar to the old DNA). This can lead to the bacteria acquiring new abilities, such as being able to grow on an antibiotic.

A whole bunch of genes and proteins are needed for transformation to happen. Sometimes, the cell doesn't even get transformed because nucleases break down the incoming DNA before it can reach the chromosome. RecJ and ExoI are two nucleases that my project focuses on. dprA is a gene that is required for transformation. It's gene product probably acts as a sort of shield for the incoming DNA against nucleases. I want to test if dprA's action is specific to either RecJ or ExoI.

So let's say we have four H.influenzae cells.
Cell #1 is a wildtype cell. It has both the dprA (blocker) gene and the one that encodes, say, RecJ (cutter). We know (from experiments, I guess) that 1 in 1000 of these cells will transform.
Cell #2 is a mutant. We've taken out the dprA (blocker) gene, but RecJ (cutter) remains. We expect about 1 in 100 000 000 of these cell to transform because dprA isn't present to protect from RecJ's action. That's way down from cell #1.
Cell #3 is also a mutant. We took out RecJ's gene (cutter), and dprA (blocker) remains. We expect about 1 in 10 000 bacteria to transform because RecJ doesn't chop up the incoming DNA.
Cell #4 is a double mutant. That means it's missing two genes; we took out both dprA (blocker) and RecJ (cutter). This one's the real test. There are two things that could happen:
A) Bacteria transform juts as well as the wildtype. dprA is specific to RecJ and not another nuclease.
B) Not a lot of bacteria transform. dprA is specific to another nuclease.

I'll have to think about a clearer way to think about cell #4...

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