Excision of foreign genes by E.coli - suspicion of attack (Feb/04/2007 )

Hi there,

I've been having trouble cloning one of my genes and I suspect my E. coli is attacking it. I'm wondering whether E. coli are more likely to excise particular foreign genes than others. What sorts of excision or even recombination machinery do E. coli have and do modern strains prevent this from happening?

Cheers, Rob.

Erm... how can E.coli attacking the gene? Perhaps they have the DNAse encoding gene? or even some RE.

is your expression strain RecA + or -?

is your product overproduced and toxic to the e coli?

and which part is giving you difficulties? the gene-splicing part, or the expression part?

OK, this is my understanding of things -
E. coli, being prokaryotic, sometimes don't tolerate eukaryotic DNA features particularly well. Secondary structures like terminal repeats, inverted repeats and other structures are often excised and removed by the E. coli proteins. There's a cell line called SURE/SURE2 which is designed to stop this from happening, I've had some success with it protecting terminal repeats on viral vector plasmids.
Also, it's common to get leaky expression from plasmids, even if they weren't designed for E. coli expression - I think mammalian expression vectors can be problematic if they have the CMV promoter, which can work in E. coli. So if the E. coli doesn't like your gene product, you get no colonies, apparently helicase and replicase genes are often toxic to bacteria.
I had some problems with what I affectionately called the construct from hell, the only cells the little **** would transform into (after ligation) was another Stratagene cell line called ABLE, which is designed for toxic constructs, it reduces plasmid copy number. But they are really expensive to buy, so it would be a pricey way to find out if they work.
What plasmid are you using?

OK, this is my understanding of things -
E. coli, being prokaryotic, sometimes don't tolerate eukaryotic DNA features particularly well. Secondary structures like terminal repeats, inverted repeats and other structures are often excised and removed by the E. coli proteins. There's a cell line called SURE/SURE2 which is designed to stop this from happening, I've had some success with it protecting terminal repeats on viral vector plasmids.
Also, it's common to get leaky expression from plasmids, even if they weren't designed for E. coli expression - I think mammalian expression vectors can be problematic if they have the CMV promoter, which can work in E. coli. So if the E. coli doesn't like your gene product, you get no colonies, apparently helicase and replicase genes are often toxic to bacteria.
I had some problems with what I affectionately called the construct from hell, the only cells the little **** would transform into (after ligation) was another Stratagene cell line called ABLE, which is designed for toxic constructs, it reduces plasmid copy number. But they are really expensive to buy, so it would be a pricey way to find out if they work.
What plasmid are you using?