RecA- strains tend to be those used to increase the copy number of the plasmid so that your transformation into a suitable strain for protein expression (such as BL21(DE3)) will be more likely to be successful. Keeping a stable, unperturbed plasmid in RecA- strains is easier. Strains designed for protein expression aren't worth going through the effort of deleting RecA as it's benefit to the purpose of the strain (i.e. protein expression) is negligible. Good practice should always use RecA- strains for long term storage of plasmid constructs and then transfer into protein expression strains as needed.

Okay, but wouldn't having an expression plasmid (or two) in a recA+ strain risk having some re-arrangement? Or is it more about keeping the copy number low, so as to not overproduce the recombinant protein(s)?

Of course the risk of recombination is there, but likely to be negligible in the time you’ve grown your E. coli for the purposes of expression. Plus, if you’re using an inducible expression system like the lac operon this is quite an energy intensive process and most of the organisms resources will be poured into expression. As RecA is an ATP dependent process for successful recombination the likelihood of a successful recombination occurring during recombinant expression is very minimal if the organisms energy is being expended elsewhere.

Okay. How about long-term expression systems, as in bioreactors? Does one then have to consider moving to a recA- strain, just as a precaution?

Hi!

A very interesting question Kiss. I feel if you are expressing a protein which has a homolog in your RecA+ expresssion strain (host) then there is certain risk that recombination can occur. But in case you are expressing a protein which has no homologs then I don't think there will be any chances of RecA mediated homologous recombination. Moreover, if the cells are healthy and not stressed by any DNA damaging factors (internal or external) then there shouldn't be any problem of HR mediated repair.

Hi Cathail,

I'm trying to express a protein in BL21 star (DE3) which has no homolog of my protein in it. But when I isolated the DNA from the expression strain (glycerol stock of the strain with the expression plasmid), and digested to see an insert release, I saw multiple bands. Do you think it is due to recombination and the plasmid I isolated from the glycerol stock might not be the original expression plasmid?

Hello again Andor,

Apologies for the late reply. Busy times. Again, recombinant expression of proteins is energy intensive. Even in a bioreactor the majority of ATP is going to be used up through expression of the protein, leaving very little for RecA mediated recombination to occur. While you could use a RecA- strain as a precaution, it is likely unnecessary and I don't think anyone has observed detrimental effects of RecA+ strains in protein expression to date.

Saha,

There are many questions I have to clarify your situation. 1) What plasmid are you using as your expression? You need to be sure that the restriction enzymes you are using for your digestion don't have sites within the reading frame of your cloned gene, this may result in the multiple banding. 2) Do you have a RecA- strain with your recombinant plasmid in it? This is best practice for long term storage and replication of your plasmid for any future expression experiments?

recA+ strains are much healthier than recA- strains, are more stable and have faster growth rates. The only time you need to use a recA- strain is if there are repeats of >15 bp in your plasmid or if there are such regions of homology between your plasmid and the genome of the expression strain.

Tias, it's a bad idea to store plasmids in expression strains as glycerol stocks. You will accumulate rearrangements and non-expressing forms especially if the antibiotic for selection is degradable by the resistance mechanism (e.g. ampicillin). The most stable way is to isolate trhe DNA from a non-expressing strain and store as a dry pellet or in deionised water or pH8 buffered solution both with at least 1 mM EDTA. For expression transform freshly every time.

Dear Cathail,

My gene of interest doesn't have the restriction sites within. I have used two different restriction enzymes to clone the gene of interest in the expression plasmid. I isolated the plasmid from the glycerol stock and did a digestion but unlike a clean insert release, we saw multiple bands post digestion.

Dear Shah,

I know that storing plasmids in expression strains isn't a good idea, but since fresh transformation of the said plasmid in the same expression strain didn't result in quite good induction, so we had to. But to our surprise the revived culture from glycerol stock did give better induction and expression than the freshly transformed cells. I get that expression strains with RecA can have rearrangements due to HR between my gene of interest and a homologous gene in the chromosomal DNA of the expression strain, but I didn't get what it has to do with the selectable marker in the vector!