I repeated the fraction-competent assay on log-phase murE749 hypercompetent cells, as I said I should in the previous post. I did a very thorough and well-controlled experiment, but the results tell me that this isn't a very reliable measure of how much the cells in the culture differ in their competence.
I grew the cells at low density in rich medium for about 3.5 hours, so they would all be growing exponentially (in log phase). I added MAP7 DNA to the cells, let them grow for 15 minutes, and added DNase I to prevent continuing DNA uptake. I then let the cells continue growing for another 1.5 hours, to allow all the antibiotic resistance alleles to be fully expressed. Then I diluted the culture and spread the cells on agar plates containing different antibiotics, singly or in pairwise combinations.
MAP7 DNA contains point mutations causing resistance to 7 different antibiotics, but I only selected for 4 of them in this experiment: novobiocin (nov), kanamycin (kan), spectinomycin (spc) and nalidixic acid (nal). The nov and kan alleles are close together on the chromosome, so I didn't select for those two together, but I selected for nov+spc, noc+nal, nal+spc, kan+nal, and kan+spc. These combinations gave me 5 different measures of fraction competent.
The nal allele gave a low transformation frequency on its own (4.9x10^-4), and all 3 of the combinations that included nal gave low estimates of fraction competent: 0.06, 0.08 and 0.09. The other two combinations gave higher estimates: 0.28 and 0.58.
That's a ten-fold range of the estimates. Practically, the difference between 0.06 of the cells being competent and 0,58 being competent is enormous, but the fraction competent assays can't tell the difference.
A former student had proposed developing a fluorescent reporter-gene assay that would let us look at cells under the microscope and count the ones that had turned on their competence genes. I still think it would be a big pain, largely because the cells are so small, but maybe the recent improvements in reporter molecules and in microscopy now make this a good idea.
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If fluorescent reporters are reliable readouts of transformation, you could use flow cytometry to count fluorescent cells -- more reliable, objective, and high-throughput than microscopy.
ReplyDeleteBut I question whether fluorescence would be a great way of measuring transformation. That assumes that all the cells that take up plasmid express fluorescence at the some kinetics and intensity. I have no experience with this for bacterial cells, but mammalian cells show a wide distribution of fluorescence intensity this way, in my experience. You might end up measuring many parameters layered on top of actual transformation.