The red system (Wanner and others) works fine in a RecA mutant. Alternately you could make the mutation using your favorite method in a RecA+ strain and then transduce in a recA allele if that is necessary for your experiment.

Not sure exactly about strain details, but you have two potential solutions. 1, supply recA from a plasmid and then to lambda recombineering or 2 (more favorable), make the mutations first then P1 out recA. Is this possible for you to do?

two good ideas. Thank you. Transduction is good idea. ... I hope I find these phages somewhere! :-)

Lambda recombineering (red-gam) works fine in a RecA mutant strain because red genes provide recombination functions.

Great thank you Michael, thank you Maira, thank you Nick!

Hey Johannes,

Michael is also correct that the beta, gam, and exo system can work for recombination in recA- strains. I have found that the recA- cells are somewhat sick and the efficiency of recombination is pretty poor, especially if your mutations make cells even more sick. That is why I favored the knocking out recA last method. Good luck making your mutants!

-Nick

This is a good idea Nick. I'll get along. Thanks a lot!

The easiest way to construct your mutant is to get a mutant strain from Coli Genetic Stock Center at Yale university. They have a set of strains from Keio collection with every single E. coli ORF knocked-out and marked with kan resistance (a cassette which can be eliminated). So you should order the strain that contains deletion of your specific allele (you will receive the shipment in few days) and then introduce that mutation into wt strain by P1 transduction. Finally, recA mutation should be introduced by P1. recA mutation can also be obtained from the same source. Good luck!

Direct one step recombineering with oligos encompassing your mutation.

Or use a double step positive/negative selectable marker such as RpsL/Neo: first select using Kan and then counterselect using Streptomycin. During counterselection you can transform with an oligo or a PCR fragment encompassing the mutation. A nice overview paper is:

Recombineering: a homologous recombination-based method of genetic engineering.

Sharan SK, Thomason LC, Kuznetsov SG, Court DL.

Nat Protoc. 2009;4(2):206-23. doi: 10.1038/nprot.2008.227.

I did it, it is able to knockout recA gene by Keio Collection and P1 phage transduction. However, since the very sick transformants and recombinase lacking with recA mutants, i had to use Red Recombinase by introducing pKD46 prior to knockout recA gene.

If the materials or strains for Keio Collection are not available in your lab now, you could do it by amplifying FRT-flanked Kan fragment from pKD13 carrying recA homologous region, and then introduced the purified fragment to the host strain bearing pKD46. I hope it help.