In preparation for some mutant-creation work we want to put into our grant proposal, I've been developing an efficient way to screen hundreds of cells for loss of transformability. We want to be able to use this method to identify cells carrying the point mutation we are engineering, after the mutation has been recombined into the chromosomes of some cells in a competent culture. Of course we can't know for sure that the mutation will eliminate transformation (finding this out is the point of making the mutant), but this phenotypic screen will complement our planned PCR screen for the mutation itself.
The mutant DNA isn't ready to recombine into cells yet (it needs to be put into a longer DNA fragment), so I'm developing my method with DNA carrying a point mutation that inactivates the recA homolog rec-1; this completely prevents recombination and thus eliminates transformation.
My initial test system is transformation with rec-1 mutant chromosomal DNA; if the desired mutant DNA isn't ready the tests can be repeated with PCR-amplified rec-1 mutant DNA. After some fumbling around I now have a screen that works.
The basic procedure is to mix competent cells with rec-1 mutant DNA, plate the cells on plain sBHI agar, and let them grow overnight into colonies, some of which will now carry the rec-1 mutation. To find these, large numbers of cells from individual colonies are then picked up with a pipette tip, mixed with DNA carrying a novobiocin-resistance gene, and transferred to sBHI agar containing that antibiotic. Colonies of normal cells will then give some novobiocin-resistant colonies, but colonies of nontransformable mutants won't.
The screen takes advantage of several things we've discovered over the years. One is the fact that many cells in an H. influenzae colony spontaneously develop competence. When the cells in a colony are lifted from the agar and mixed with DNA carrying a selectable mutation, some of the cells take up the DNA and acquire the mutation.
For wildtype cells the frequency of this 'colony transformation' is quite low if the mutant DNA is the chromosomal DNA of a mutant cell, because most of the DNA the cells take up comes from other parts of the chromosome. But the transformation frequency is much higher is cells are instead given a pure DNA fragment carrying the mutation, either a cloned gene or a PCR product. We have a cloned novobiocin-resistance allele that transforms very well.
We also can greatly increase the frequency of this transformation by doing the initial transformation of our candidate point mutation into hypercompetent cells rather than normal cells. Because 100-times as many of the cells in the colony are competent, we don't need to plate a large number of the cells from each colony in order to distinguish colonies that give transformants from colonies that don't. Instead we can just put a small spot of cells from each colony onto the antibiotic plate. Because we know that the hypercompetence mutation only affects regulation, using these cells won't compromise our ability to detect effects of our engineered mutations on the uptake machinery.
One other minor but very useful thing we know is that some antibiotic-resistance mutations don't require 'expression time'. With most resistance mutations, cells that acquire the mutation by transformation take an hour or more of culture without antibiotic to express the proteins that will now make them resistant to the antibiotic. But this expression time isn't needed for novobiocin and kanamycin - instead cells that have taken up novR or kanR alleles can form colonies even if they are immediately placed on antibiotic agar.
In my optimized procedure, each colony is picked up with a pipette tip, and the tip is touched to a sBHI plate to preserve the colony in case it turns out to be the mutant we're looking for. The rest of the cells are then quickly suspended in 50 µl of sBHI containing 1 ng of the novR DNA fragment (in a well of a 96-well microtiter plate), and 5 µl of that is immediatelyspotted onto an sBHI+novobiocin plate (16 spots per 60-mm plate). After overnight incubation almost all the spots contain hundreds of colonies - the rare ones that don't contain colonies are the desired mutants.
This screen is fast and efficient enough for our needs. In a few hours I can test several hundred colonies - since we expect our engineered-mutant DNA fragments to transform at better than 1%, this should be enough to find each mutant. In the first test I found one rec-1 mutant colony among the 64 I screened.
Aug. 29: In a replicate experiment I've screened another 144 colonies and found 3 mutants.
Welcome to the 4th Reich part 1.
6 hours ago in Angry by Choice