Cloning gene of interest in ecoli

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Cloning gene of interest in ecoli

Postby dummykg » Nov 14 2016 11:26 pm


This is a quick question. I am new to the field of molecular Biology. Recently I inoculated a colony of ecoli that I obtained after transformation in 4ml of Luria broth media for 16 hours, did a miniprep using 3ml of the media and sequenced the DNA. The sequencing result is positive and it is confirmed that my gene of interest is present in the vector. My question is, how can I sure that every ecoli cell in the remaining 1ml of the media has the same positive sequence. Do mutations happen when ecoli replicate?

Thanks in advance
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Re: Cloning gene of interest in ecoli

Postby relaxin » Nov 15 2016 1:05 pm

Yes, mutations do occur in E. coli. That is why we always grow plasmid prep from a single colony. However, the mutation rate of the E coli hosts we normally usedis quite low. Unless the cloned gene is toxic to the host growth, mutation or deletion of the gene is generally not a problem.
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Re: Cloning gene of interest in ecoli

Postby r.rosati » Nov 17 2016 10:19 am

Point mutation is a very rare event. Possible, but as Relaxin wrote, if there is no fitness advantage to the bacteria in the mutation, then its chance is usually negligible.
Recombination is usually only a problem with very large plasmids that contain homologous sequences (say for example, a cosmid, or a PAC, or a BAC containing genomic human DNA with multiple ALUs). in this case the recombined bacteria might have a growth advantage by not having to dedicate resources into duplicating the whole plasmid. Many strains are RecA- so they will recombine less frequently; but recombinaton by RecA requires a few hundred nucleotides of homology, so this is not an issue for most cloned cDNAs.
Elimination of the plasmid altogether occurs when selection pressure is reduced; i.e. with ampicillin, when bacteria start producing enough beta-lactamase that the medium is depleted of antibiotic. this however does not result in mutation per se; it just results in little or no plasmid being recovered.
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