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一道非常考基本功的题。
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在某主页上看见一堆分子生物学的题，好考基本功啊！ 这题看了答案也没想明白，各位看看题讨论讨论。 It is possible to obtain mutations in plasmid encoded genes by mutagenizing the purified plasmid DNA in vitro (for example, with hydroxylamine) then transforming the plasmid DNA into recipient cells. Given a plasmid that encodes ampicillin resistance (AmpR), chloramphenicol resistance (CamR), and B-galactosidase (lacZ), how would you use this approach to isolate single mutations in the lacZ gene? Be sure to describe any genetic selections and/or screens used. [Hint: you will need a way of measuring the relative amount of mutagenesis to ensure a reasonable probability that the lacZ mutants are due to a single mutation.] ANSWER: Mutagenize the purified plasmid DNA in vitro with hydroxylamine. Move the plasmid into a lacI- lacZ+ lacY+ host (by transformation or electroporation of the plasmid DNA), selecting for chloramphenicol resistance. Screen the cells for (i) AmpR and (ii) repression of lacZ (for example, by screening for X-gal- in the absence of an inducer such as lactose or IPTG). In several independent experiments, quantitate the extent of mutagenesis by plotting the number of Amps mutants and the number of LacI- mutants vs time of exposure to the mutagen. The results will indicate the number of cells that have a mutation in both genes (i.e. at least two hits) compared to the number of cells that have a mutation in only one gene (i.e. at least one hit). These results can be plugged into the Poisson distribution to determine the probability of 0, 1, and >1 hit per gene for each time point. Choose colonies from time points where the probability of >1 hit per gene is rare. [ Last edited by 1949stone on 2013-6-6 at 08:08 ] |
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