I am working on knocking out a gene from Acinetobacter baumannii (gram -ve) and need a counter-selectable lethal gene like sacB, but do not have any specialized vectors for that. I am using forward and reverse flanking regions of the gene interrupted by Kan marker as the knock-out cassette and cloned into pUC19 and pJET (unable to replicate) both. What I get is only integrants (single recombinants), they are able to grow in ampicillin when counter-selected (plasmid backbone has ampR marker, a double recombination indicates a plasmid loss, so not able to grow in amp). The colonies are thousands in number which makes a tedious job to plate them onto ampicillin-LB plate. Therefore, will just amplifying and cloning the sacB gene from B. subtilis genomic DNA will be enough to get a suicidal vector?
No, it won't work. You need a vector with R6K origin of replication and harboring sacB gene. Two vectors I have worked with are pWM91 and pCVD442.
Thanx Syed. In fact, the plasmid not having an origin of replication is fine for me. (if I am not wrong) The time the plasmid spends in the cell is sufficient to get itself integrated (I am getting colonies with pUC19), but the difficulty is in isolating double crossover mutants. If the plasmid is unable to replicate, that will make me easy to get a mutant with a loss of the plasmid. Please guide if I am wrong.
Yes, you are right and plasmids with R6K ori do not replicate in non lambda pir hosts hence used in suicide plasmids. You are using pUC19 and pJet, both, to the best of my knowledge, contain pMB1 ori and therefore I am unable to understand how you get integrants (merodiploids)?
What is the length of the gene flanking the Kan cassette? The medium you are using, it contains salt? At what temperature you are incubating cells?
Correction... Yes, ColE1 based plasmids do not replicate in Acinetobacter baumannii and there both pUC19 and pJet can be used as suicide vectors.
Thanx Syed. With this, I am assuming that I will get a clone of pUC19-SacB or pJET-SacB and clone the Kan cassette therein. The length of my cassette is Forward-Kan-Reverse = 312+937+313 = 1562. DNA is eluted with Nuclease-free Water. Incubate cell at 37 deg C. After getting integrants, I subculture them in LB broth with and without selection to allow plasmid loss. Any more suggestion/improvements regarding isolation of the double-crossover will be helpful. Again, thanks for taking time for this conversation.
I am afraid that flanking sequences are not long enough. Homologous recombination works best with 500 or so bases, in fact, I prefer 700-1000 bases on each side. Secondly, the culture medium should not contain NaCl and sacB expresses best at room temperature. Give these suggestions are try.
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