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Stability of plasmids in recA1 strains - (Oct/19/2007 )

I am a bit confused:
When reading the GEX manual from GE Healthcare, it is said, that for cloning one should not use "an E. coli strain carrying the recA1 allele for propagation of pGEX plasmids. There have been reports that these strains can cause rearrangements or deletions within plasmid DNA."

Instead JM105 is recomended which does not have recA1.

Until now I thought that recA1 is better for cloning and maintenance of plasmids as it reduces occurrence of unwanted recombination in cloned DNA. That´s why people are using strains like DH5a, JM109, XL-1blue, TOP10, etc. for cloning.

Of course GE hotline was not helpful in this case..Wink

Can anybody explain the difference in my understanding and the GEX manual please.

-Dukes-

recA minus strain is what you are thinking about. Wild type E coli have functional recA genes. recA protein is use for repair work by coating single stranded DNA and mediate DNA recombination. If you look carefully all the strain you have listed are recA1- stains. The gene has been knocked out, hence it is listed on the cell's phenotype.

-perneseblue-

QUOTE (perneseblue @ Oct 19 2007, 10:44 PM)
recA minus strain is what you are thinking about. Wild type E coli have functional recA genes. recA protein is use for repair work by coating single stranded DNA and mediate DNA recombination. If you look carefully all the strain you have listed are recA1- stains. The gene has been knocked out, hence it is listed on the cell's phenotype.


I guess I haven´t expressed it clear enough, sorry for that.

Of course I meant with "having recA1" that the strains have the mutation of recA1 listed in their phenotype and by this are recA1 minus. And I know that DH5a, JM109, Xl-1 blue etc. are recA1 minus. As I understand it, they are routinely used because of this mutation for cloning because they cannot recombine the plasmid.

But in the GEX manual it is explicitly said NOT to use those strains but rather JM105 which is recA1 native. Why?

-Dukes-

QUOTE (Dukes @ Oct 22 2007, 08:03 AM)
QUOTE (perneseblue @ Oct 19 2007, 10:44 PM)
recA minus strain is what you are thinking about. Wild type E coli have functional recA genes. recA protein is use for repair work by coating single stranded DNA and mediate DNA recombination. If you look carefully all the strain you have listed are recA1- stains. The gene has been knocked out, hence it is listed on the cell's phenotype.


I guess I haven´t expressed it clear enough, sorry for that.

Of course I meant with "having recA1" that the strains have the mutation of recA1 listed in their phenotype and by this are recA1 minus. And I know that DH5a, JM109, Xl-1 blue etc. are recA1 minus. As I understand it, they are routinely used because of this mutation for cloning because they cannot recombine the plasmid.

But in the GEX manual it is explicitly said NOT to use those strains but rather JM105 which is recA1 native. Why?


Ah my mistake. Sorry.
Nope no idea. JM105 is also recA1 plus. So it is pretty bad for maintaining a plasmid. Perhaps it was a typo? JM108 and JM109 are recA1 minus.

-perneseblue-

E. coli has a repair system which will recombine homologous sequences. Many people fear that recombination can cause plasmids to rearrange or delete insertions because of recombination. For this reason, cloning strains are traditionally recA mutants. RecA strains have the advantage of having simple plasmid profiles (mostly CC monomer) whereas Rec+ strains have dimers, trimers, etc. and their nicked relatives that make uncut plasmid lanes complicated.

Genomic clones often have duplicated regions, but these duplications are usually rather short,tandem duplications. They are unstable, but this instability is not due to recA function. If the length of the duplicated sequence is less than 200 bp, recA has no effect whatsoever.

The recA repair system is useful to E. coli and disabling it causes the cells to grow slower and be less healthy. For this reason, some expression strains (like BL21) are not recA mutants.

So this could be one reason for JM105 being recomended RecA+ strains as they are used for expression as well.

-cloned-