Lab update

I’ve been trying to find the transformation frequency of  my DprAcomM mutant. This has been very slow and frustrating work, mostly due to the fact that it’s the combination of two mutants known to have low transformation frequencies. I’ve had to alter my experiment many times to adjust to the unique difficulties of working with this strain, plating very large numbers of cells to ensure I get some transformed colonies, over enough agar to not have any issues with overcrowding, and controlling for everything to make sure I can back up those statements. Good No DNA controls are essential since there is some rare background resistance mutation to my antibiotic of choice, Nov. Still no clear answer, although it’s obvious the transformation rate is low. At this point I’m shelfing this strain, and instead starting work on the other mutants I need to find transformation frequencies for. They should be much easier to test, and when I return to ComMDrpA hopefully I’ll have cleaner experiments.

 

I’m currently doing a small investigative side project based on an observation I made a few weeks ago. I had a large batch of antibiotic plates containng DprAComM transformants from the previous days experiments, but since the colonies were so small and rare, and the colonies were harder to discern from the cruddy plate background since they were inncoulate with such a high density of cells, it was very hard difficult to get an accurate unambiguous count. So I threw these plates back into the 37 degree incubator over the weekend, hoping the borderline colonies I was tenative about would be larger and less ambigous. The following Monday I found many times more colonies appearing on the plates than I had expected, almost a magnitude. Initially I thought this was an effect of plating at such a high denisty – some type of growth inhibition due to density. I’ve been recounting my plates since, the day after plating as I’ve always done, then putting them back in the incubator and additionally counting after 48hours. What I’ve found is that while the plain plates have the same number of colonies, the counts for the antibiotic plates consistently increases significantly, for all strains including wildtype.

I can think of 3 possibilities:

1) This is an effect of the antibiotic slowing growth. Very likely, since colonies on antibiotic plates appear smaller than colonies on plain plates.

2) This is an effect of cell density. Antibiotic plates always have several fold more cells than plain plates

3) This delayed growth is a property of transformed cells. There is a relationship between competent cells and persister cells

To test these possibilities I need a controlled experiment, not just observational. I’m planning to grow up Nov resistant KW20 cells and plate them at equal densities on antibiotic and plain plates, taking colony counts over a few days. If there is no difference between the antibiotic and plain plates I can rule out the antibiotic as the culprit. I currently have an ON culture of Nov-R cells taken from a plate today growing in the lab, since we never keep Nov resistant cells. Testing the effect of densities will be more difficult.

I’d like to test this effect longer than a few days but our bact requires fresh vitamins to grow, after a few days the hemin degenerates and the colonies die. Adding new hemin onto the plates would spread bact around founding new colonies.

 

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