Hello,
I want to take a timeout from my other project and now I am into genetically engineered bacteria. I want to use the pje202/pVIB plasmid containing e. coli. This bacterium has to grow on ampicilline or it will eventually lose the plasmid. I wanted to ask, whether there is another possibility to prevent plasmid loss other than using antimicrobial drugs?
Thanks very much,
regards,
André Leonhardt
Hi Andre,
There are a number of options for antibiotic-free plasmid retention in E. coli. The most commonly used methods involve knockout of an essential gene from your host E. coli, which is then complemented by including that gene within a plasmid. As the E. coli will die unless it contains the plasmid carrying the essential gene, you will prevent loss of the plasmid.
Please refer to this review article for further details: Article Antibiotic-Free Selection in Biotherapeutics: Now and Forever
Microbes are fighting against other microbes in order to get hegemony.
Benzylpenicillin (Penicillin G; PCG) is produced by fungus to kill bacteria. Ampicillin is produced by industrially aminating PCG. Bacteria can fight to Ampicillin by using beta-Lactamase. beta-Lactamase gene may be donated by symbiotic bacterial virus or bacteriophage or plasmid.
Bacteria (Lactobacillus casei Shirota) has already been infected by bacteriophage and/or plasmid at 12% of cell protein (our unpublished observation).
I have searched for weapons in a bacterium of Lactobacillus casei Shirota.
Wall-associated protein, which is a ribonuclease (RNase) against single-stranded RNA bacteriophages, at 46.8 μg/mg of cell protein.
Lethal factor, which is a metalloprotease and may hydrolyze proteins of bacteriophage, is present at 9.5 μg/mg of cell protein.
Pesticidal crystal protein Cry4Aa, which is a pesticide protein, at 9.1 μg/mg of cell protein.
Therefore, invaded immigrant plasmid or bacteriophage may be defeated in the battle against host bacteria and/or host endogenous bacteriophage.
Just a bit of an addition, if you wish to preserve the above strain of E. coli with that particular plasmid, there is no other way to prevent the plasmid loss or plasmid curing unless you keep the selection pressure on the strain.However, there is an alternative although not very preferable, what you can do is culture the first primary culture in the presence of the antibiotic and make several glycerol stocks of it (the number depends upon the number of experiments you wish to carry with no antibiotics). Once you have made the glycerol stocks, you may use the fresh set of bacteria every time you need it. But do bear in mind that this increases the chances of not only extra contamination in the culture as there would be no selective advantage for the bacteria containing the plasmid since the strain without the plasmid would grow faster than the strain carrying the plasmid but also the chance of having mix culture of bacteria as in those with the plasmid and those without the plasmid, the only difference here is that the contamination rose from existing strain of bacteria. Hence, unfortunately, unless you modify the bacterial strain or change the characteristics of plasmid, I find it slim chance of preserving the plasmid without the selection pressure..
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