Hello,
I recently bought a genetically modified e.coli (pje202/pVIB plasmid), which is bioluminescent. I don't want to grow the e.coli on antibiotic agar forever, because the antibiotic selection has many limitations. The plasmid produces b-lactamase, so it is resistant to f.e. ampicillin, carbenicillin etc.
If I don't grow it on lb-amp, it will lose its plasmids. I wondered, whether I can slow down the plasmid loss, by growing the bacteria on drug-less lb agar, then isolate the brightest colony, regrow it on lb-amp and then again grow it on pure lb and isolate again. I thought, this way I may isolate the mutants that are less likely to lose plasmids, because bacteria containing the plasmid will glow only.
Thanks very much,
regards,
André
Not sure what you are asking. Are you asking to select for brighter colonies? if you are then putting the on no selection is not going to do it. The bacteria will just kick out the plasmid regardless. If you don't want to continually culture the same sample, each time you need to use it just go to your frozen stock and re-streak the culture. I am not sure what you mean by antibiotic selection has its limitations. Limitation is what you can do? it shouldn't, even if you need to do an assay that can't have antibiotics in it, you can grow the bacteria in antibiotics, the spin it down and use it. It will glow and unless you need to use it for an extended time, it should glow for a while
I am with Victoria. I don't see a reason not to use an antibiotic. If the test itself really needs to be without antibiotic, you transfer the bacterium at the moment of starting your last incubation. Plasmid loss does not go that fast and happens at random throughout the culture.
I want to create a bioluminescent ambient lamp, which can use old (sterilised) food waste. I would grow the e.coli pje202 on amp-lb, but the lamp should glow a long time. The ampicilline degrades readily after 12h, so it would have to be kept cool.
I thought
lb-amp = plamid-e.coli
lb = most darker colonies, because of plasmid loss.
I thought if i isolate the brightest colonies from the pure lb, I may get mutants that are less likely to lose plasmids, because the brightness may indirectly indicate the yet high plasmid content e.coli.
So your goal, if I understand correctly, is to create a strain of bacteria that continually express a transgene? If so, maybe using a plasmid is not the best option. You might want to consider a stable integration in the genome e.g. by lamda-red recombination. Selecting the transformants will be very easy as colonies should be luminescent. Also consider using a selective mechanism (e.g. toxin-antitoxin or something similar) to prevent gene inactivation on the long term.
Hope that helps
Bacteria are very efficient organisms. As a general rule of thumb, if they don’t have a reason to keep the gene they will lose it in favor of keeping essential genes, which is why they lose the plasmid if antibiotic is not present. They have no reason to keep a resistance gene. They will ditch the fluorescent gene, too, if it’s not expressed. I agree with Raphael, a stable integration into the genome is better. If you’re developing a product that has live microbes in it you don’t want them to have antibiotic resistance.
The brighter colonies may be expressing more of the fluorophore (fluorescent protein) than the darker ones. There can be many molecular mechanisms of why this is so. There could be random mutations making the promoter stronger, or multiple insertions of promoters and/or gene in plasmid or copy numbers are different among colonies, other mutations in colonies are expressing unknown repressors of expression or dampen fluorescence. For practical use of your time, you might get some information by sequencing some bright and dark colonies to check for mutations, checking quantity protein expressed.
If you grow colonies without antibiotic and still succeed in finding fluorescent ones, some of these may possibly already have the gene inserted into genome (presuming all of them lost plasmid). You can quickly test by pcr where it is and whether it is still in a plasmid or not. You have to restreak best candidates many tines to ensure it is stable for your purposes.
Good luck!
I need to disagree with much of the above advice. Plasmids are often quite stable and hard to lose. When I've tried to "lose" a plasmid from a strain, it can be very difficult to do and require lots of screening. So it is likely that you can grow your strain for a many generations and passages without adding selective drug and most the cells will maintain the plasmid. HOWEVER, some plasmids impose a significant metabolic burden and those are selected against. So it very much depends upon the plasmid itself. You can test very quickly, just grow an overnight culture without ampicillin, plate for single colonies and test a 100 colonies or so either to see if they retained drug resistance or if they glow.
But I agree that there usually is not any reason to not use ampicillin in the medium.
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