I am working with seven strains of Keio collection, but I have hard time to remove Kanamycin marker from one of those, particularly the one with Delta TyrA. I simply removed kan from the rest six by pcp20 but after several attempts I have not be able to remove kan from delta TyrA.
I asked the CGSC to send me a new one but even that did not work. I changed pcp20 to pE-FLP but still could not remove the marker. I used the P1 transduction to construct the double knockout, it was successful (phenotype and PCR check) but again I can not remove Kan from this strain.
I appreciate any suggestions.
It may be that the strain from which you are unable to remove the Kan marker has undergone some adjacent deletion or rearrangement that removed one of the FRT sites. You could confirm this by DNA sequencing if it is important, or you could just work around it by building a different deltaTyrA strain.
Hi Mahamoud, did you ever find a solution to your problem? I'm facing the same thing.
Sammy,
No! so we decided to use lambda-red system (pKD46) to create the KO for TyrA .
But using lambda-red will also insert a kanamycin or chloramphenicol marker. Was pCP20 more effective in this case? Maybe you knocked out a different part of the genome than is present in the Keio collection?
We KO tyrA in parent strain BW25113 using the same primer in original paper and Cm marker. There was no problem to remove the Cm in this new construct using either pcp20 or pE-flp. PCR check and sequencing confirm correct removal of tyrA.
@ Mahmoud Kamal Ahmadi Can you please share the protocol? as I am also facing the same problem
@ Sammy Pontrelli were you able to solve the problem?
Hi @Mahoud Kamal Ahmadi,@SalmaWaheed
I am facing the same problem. Some of the strains I am using are not totally have transformants with pCP20 plasmid while there are colonies with any other control plasmids. Is your problem is the same (no transformants)? Or you are getting transformants but kanamycin cassette is still in there (Kan resistant) after ON incubation at 42OC?
I have this problem too, the kanamycin cassette is not removed by PCP20 in one of my mutants.
Could do a sequencing to check the FRT sites, any other suggestions?
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