Friday, July 17, 2009

Dose Response

How hungry are competent cells for DNA? I know that about a billion cells will consume ~65% of 20 nanograms tasty USS-1 fragment, but what if I offer the cells different amounts of USS-1?

To get a better hands-on feel for the DNA uptake process in wild-type and rec-2 mutant competent cells, I did a dose response experiment, where I incubated competent cells with different amounts of USS-1 DNA.

For this first experiment, I used 0.5 ml of competent cell cultures for each sample and did 6 different amounts of USS-1 DNA (12 samples total for wt and rec-2). I didn’t have enough radiolabeled fragment for all of my desired concentration, so I mixed in some cold USS-1 DNA to make up the difference. I let the DNA and cells incubate for 30 mins, then I washed the cells several times and determined the total radioactive counts in the cell pellet and washes to determine the % uptake and total uptake.

Here’s the results:

Total Uptake:

Percent uptake:

Interestingly, rec-2 does better at low concentrations of DNA than wild-type, but worse with high concentrations. The latter could be due to the periplasm getting too clogged with DNA, such that the outer membrane uptake machinery has to work too hard to get more DNA through, while in wild-type translocation of DNA frees up space in the periplasm. But the former (higher uptake in rec-2 at low DNA concentrations) doesn't really make much sense to me. Maybe not all free nucleotides created during degradation at the inner membrane remain in the cell, so that at low concentrations, rec-2 simply holds more label?

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