The resifual construct that you used to prepare the KO may be causing some odd recombination with your transformation plasmid that may be causing deletion of your selection marker. You can try to overcome this using old school calcium chloride transformation. If that does not circumvent the issue, you will likely need to use a different selection marker in your plasmid.

One possibility is that you have a contaminant strain and what you think is your KO is actually something else.

I would suggest designing PCR primers that will serve to diagnostically verify your final genotype construct is what you presume it is and also show that the material lost after homologous recombination is actually gone.

Are you electroplating a new replicating plasmid or are you relying on homologous recombination to see transformants? If the latter, it might be you have a deletion or rearrangement of the target region so the problem is the recombination event.

- Are you sure about the concentration of the antibiotic you are using (after electroporation), are you putting too much of antibiotic?

- suggestion:

**Culture the electorate cells on growth medium free of antibiotic to see if the problem is about antibiotic and resistance gene or sth else that occurs while implementing the electric shock.

**Use another plasmid with different resistance gene...

Michael J. Benedik

Thank you. I performed genotyping PCR with primers than span the deletion. The two clones I selected yielded a small band consistent with the deletion genotype. Other candidate clones yielded a large band consistent with a WT genotype.

The KO strain shows no hygromycin resistance, which I think is definitive the suicide plasmid (which was used to make the deletion) is gone.

The plasmid I am electroporating integrates into a tRNA gene. It can be electroporated into any strain of this species that does not already have the plasmid integrated at that site. The gene I deleted should have had no effect on the tRNA gene or plasmids that integrate into the tRNA gene. I verified by PCR that my KO strain does not have a plasmid integrated into this tRNA gene (I had positive controls in parallel and they showed a band consistent with plasmid integration).

Overall, the color of the culture, the doubling time, and colony morphology looks all normal for this bacteria.

Saber Delpasand Khabbazi

The concentration of antibiotic worked well for WT and the transposon mutant. I electroporated WT and my KO strain in parallel with the same plasmid, and plated on the same plates. The result was: WT showed hundreds of colonies, my deletion strain showed none.

I will try to test the growth on antibiotic-free plates, however I think it is unlikely they died during some part of my electroporation process. I initially thought that might be happening, so I carefully observed my electroporation process. After electroporating, I recover for ~16-24 hrs in antibiotic free media, then pellet the bacteria before plating . The pellet size was large and the same size between my deletion strain and WT strain. Bacteria that have been dead for a long period do not pellet well in my experience, so I suspect there are a similar number of live bacteria between WT and my deletion strain. However, it is true this is not definitive and would require more testing.