Hi Giorgia,

Unfortunatelly I don't know the pKD46 system. It can be an off-topic answer, but maybe this very powerful alternative can get a chance. I've heard CRISPR Cas9 system to use gene editing from prokaryotes to human. Probably you have heard about too. Here you can find an attached rewiew about this system. Anyway I need to read about this decent and powerfull system.

Bests, Tamás

Yes, the system should work in these strains of bacteria. Before you question the efficacy of the system in these strains, did you see if you could get it to work in other strains?

As as for a protocol, I just followed directly the same protocol published in the source paper that developed the system. A couple tips: for some reason if I use kanamycin solution that was stored in 4c and not -20c, it doesn’t work. Also, some people have noted that if you induce the cells (with arabinose) overnight then you will get increased efficiency. Also make sure you designed your primers correctly.

Seconding Sammy Pontrelli's answer. Also, don't use CRISPR Cas9 for editing E. coli, it's absolutely not worth the trouble.

@Sammy Pontrelli I always use kanamycin stored in -20. I've been using this protocol for DH5alpha harboring pKD46:Article One-step inactivation of chromosomal genes in Escherichia co...

and I've noticed the difference between using 1mM of Arab and 10mM in growing the cells. The cells overnight are more efficient using 10mM of Arab. This morning I got the clones and I should follow the final steps. The real problems is how I should design the primers to get the sequence on the genome where my gene has been ko.

I've been trying (using other systems) to design primers before the sequence of my target but they all seem unspecific so I always failed.

Any good advice for that?

@Alexandra Jonhson yeah, I actually tried to use the coupled system Cas9 and pTarget but somehow it doesn't work on BL21. I also use another strain harboring lambda red and so far seems a good and rapid method.

Now I just need to overcome the sequencing problem.

Thank you all for your precious answers!!!

@ Giorgia Croppi..please let us know did you solve this problem...because i am also facing same satiation..

Something to consider when using pKD46 in an Ara+ background like BL21 is that if you add the arabinose too soon when making the competent cells, they will consume the arabinose and you will get reduced expression from the pBAD promoter on pKD46.

This is one of the reasons why the Keio knockout collection was made using the Ara- strain BW25113 as a genetic background. Since the strain can't metabolize the arabinose, it acts as a gratuitous inducer for the pBAD promoter controlling the expression of the Lambda Red genes on pKD46.