RecA deleltion - (Jun/29/2008 )

I was wondering if deleting the RecA gene from the E.Coli Genome is trivial, and can be done easily? Are there, or does anyone have a idoit's guide to this or should this be done by a more competent/skilled microbiologist?

I normally do a lot of molecular biology, but my lab doesn't work with phage (which I think this would require) and was wondering if it would be possible with some kind of cellular mating of some kind.

You could easily do this with Lambda RED or recET recombination, no phage (directly) involved, just some phage genes on a heat inducible plasmid. Given the large number of recA minus strains around, why do you need to do this?

Our lab has obtained an e coli strain that we are using in a selection, and apparently when we transform this strain with our library we are unable to isolate plasmid out of the colonies. We have even tried isolating the genomic DNA, and tried pcr to see if the genes were incorporated into the genome to no avail. Sometimes we get shorter than expected DNAs when we isolate plasmid, but more often we get nothing. So it may be that RecA may be responsible, since it is not known if the strain we are using is RecA deficient (Actually, I've just recently contacted the lab that constructed the strain, and it is NOT RecA-). So, I'm optimistic that this well help in the acquisition of plasmid after colony isolation.

I think this is unlikely to be your problem. What strain are you transforming? More likely you have an endA+ strain and are losing DNA after extraction. Make sure to do the extra washes and to work quickly during the miniprep. Why do you need to get your plasmid from this strain? What is special about this strain that makes you want to use it? Check the antibiotics -- you may be losing the plasmid during growth, if your selection is not good.

We need to get our plasmid from this strain because the plasmid that contains the gene of interest is supposed to supplement the growth to the e. coli (its an auxotrophic strain). As far as cleaning up the DNA, I normally clean up the plasmid fairly quickly (I feel; <15min total), and include the additional washes (we use Qiagen). When we grow up the colonies, the majority of the time they do not grow with the antibiotic(lose of plasmid?), and so we grow them in non-selective conditions (but they do grow on selective agar plates). The plasmid is a low copy number.

If it won't grow with antibiotics, it has lost the plasmid, and you will have trouble isolating it (as you see). Your plates may not have sufficient antibiotic to select colonies with plasmids. Make sure you are adding antibiotic when the agar is sufficiently cool. Don't grow cells for more than 12-16 hours on amp only plates, else satellite colonies without plasmids can form. You might want to do a control with non-transformed cells on your antibiotic plates to make sure they don't grow. Also worth checking -- if it is a low copy plasmid, then perhaps you are adding too much antibiotic to your liquid medium, and preventing growth.

We've tried the possibilities that you've listed. I wonder if there are ways of increasing the probability of retaining the plasmid such as lowering the growth temperature?