We know that when we use prokaryotic cells such as E. coli to express proteins, we often transform the E. coli with a plasmid containing the target gene, then screen for positive clones through antibiotic resistance, and propagate them in a medium containing antibiotics. For the positive clones that have already been screened, how long can the plasmid persist under conditions without antibiotic pressure, and how quickly will it be lost? Thank you for your answer.
Hello,
Several factors can influence the rate of loss of a plasmid in the absence of selection pressure, including: plasmid stability or replication-transcription conflict.
To avoid all of these, always maintain selection pressure by adding the antibiotic to bacterial cultures.
There is a citation from work MATHEMATICAL MODELING OF THE EXPERIMENTAL EVOLUTION OF TRANSGENIC BACTERIA: “PLASMID PARADOX”Article MATHEMATICAL MODELING OF THE EXPERIMENTAL EVOLUTION OF TRANS...
"The "plasmid paradox" lies in the fact that although the presence of plasmids containing cloned genes usually reduces the specific growth rate of transgenic bacteria, but with prolonged growth of bacteria under non-selective conditions ("experimental evolution", 20-30 or more generations of the original form without antibiotics) “cost of fitness” is reduced by an order of magnitude or two. An analysis of the experimental results leads to the conclusion that this is due to a change in the difference in the growth rates of plasmid and plasmid-free cells, with a change in the probability of formation of plasmid-free cells, the copy number of plasmids, and the expression of cloned genes in cells of transgenic bacteria under various environmental conditions."
In simple terms, plasmids introduced into E. coli can drop below 5% during growth in the absence of a selective factor after 20 to 120 generations, according to the experimental data. Interestingly, there have been a couple of cases of simultaneous elimination of native plasmids from strains cultured in three laboratories located in different countries.
It is true that some plasmids are lost at some easily frequency but other plasmids are very stable and in fact are extremely hard to lose. It depends upon how deleterious or burdensome the plasmid is to the cells. When you have a condition where the plasmid is over expressing a protein then often it will be lost (or accumulate mutations) quite rapidly because the selection is strong. But non-expressing plasmids are often quite stable. I've done some experiments and less than 1% of the cells have lost the plasmid after about 20 generations. So it all depends.
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