I recently bought pVIB plasmid modified e.coli (BL21 strain) and now I want to grow them on LB agar containing ampicillin for selective pressure. I wondered, whether there is a chance due to spontanous mutations that a non plasmid containing e.coli might survive on the amp agar?? Could it ruin my culture?
What can I do to prevent it?
Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one or more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
Basically what Artur said:
Be sure to have the original bacterial stock just in case something goes wrong. From this original stock generate one or more secondary stocks which you will actively use.
Additionally, keep an isolated plasmid stock as secondary fall-back option, so you can potentially transform a fresh E. coli stock (or even another species/strain for other uses).
Also remember mutation rates leading to spontaneous resistances are extremely low, so it is unlikely you will encounter this problem. As mentioned by Artur a loss of plasmid or loss of function are far more likely.
It is also recommendable to renew your agar plate cultures regularly from your frozen "secondary" stock. This is not only to decrease the chances of mutations, but also since the bacteria will adapt to growth on agar after some time, increasing lag time and potentially decreasing growth rate in liquid cultures.
Finally, when producing a new "secondary" stock it is always a good idea to isolate the plasmid from the same culture you used to generate the stock in order to make sure the plasmid is still present.
Hi,
for the BL21 strain, being recombination proficient, it is very well possible that a part of the plasmid (possibly only the resistance gene) is integrated into the genome, yielding an selection-resistant strain possibly without the other features present on the original plasmid. This is why for pure DNA work, usually recombination-deficient strains (e.g. DH5 alpha) are generally preferred.
Apart from that, I agree with Mathis, when in doubt fall back to clean stocks (non-transformated competent BL21 cells) and do a fresh transformation with your prepared plasmid.
If you want to be absolutely sure on the presence of your genes on a plasmid (and not in the genome), perform clean plasmid preps (no colony PCR, as it will be positive for genomic integration, too!) on the obtained clones and screen those by PCR, however for routine transformation this would be quite an overkill in my opinion.
Previous responders all have excellent advice for good practices. I have worked in both molecular bio and microbiology labs and yes, E. coli can and do acquire mutations in the genome that confer antibiotic resistance and acquire contaminant plasmids from other lab projects that have resistance but not your insert. Happens infrequently, but more often than you would like, and can cause loss of days, weeks, even months of wasted time.
Best way to be sure is to isolate plasmid from a few colonies, as suggested by Patrick. I would do restriction digest or pcr to confirm. Just having prescence of plasmid DNA doesn’t guarantee it‘s the one you want. E. coli can acquire plasmid on their own and may have done this in the resource lab producing your stock. These could be antibiotic resistant.
Once you confirm you have the one you want, you can go back to the overnight and make glycerol stock to freeze. This is the only time you should have to do all this checking, unless a question comes up again.
Keep in mind that pcr is notorious for amplifying contaminant dna from lab sources like pipettes, researcher hands, bench, atmosphere, etc. So you need very good practices to be sure your pcr result is valid.
Good luck!
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