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.
For the last few weeks, the blue text of your posts has appeared "behind" the almost opaque green background and has been practically unreadable--to this Mac OSX/Safari user at least.
ReplyDeleteCan you fix it, please?
good post. Relps me by writing my thesis
ReplyDeleteHi Rosie,
ReplyDeleteit is always great to read your blog.
Do you have cell division during those 1.5 hours after adding the map7 DNA? In which case perhaps frequencies are not very accurate and you would need a Luria-Delbruck kind of analysis for more precise estimates? Perhaps one that thakes into account that some resistance genes cause changes in growth rate.
Lilia
Hi Rosie,
ReplyDeleteLong time, no post. Is this blog dead now? I hope I everything is okay and that you'll start writing again.
In any case, I just wanted to say that I've really enjoyed reading your blog over the last couple of years and that it has been really useful and insightful for me as someone who's just now starting a PhD.
Your research about combining them is look awesome, I don't really knew about DNA. But your research make me understand how difficult to be scientist to create something useful for human life. I will appreciate your research this means something for me.
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