gene disruption - should regions flanking a cassette be of equal size (Jul/10/2007 )

Hello all,

I have to disrupt a gene cluster (nifH-D) in a bacillus like bacterium of which very little sequence information is available. I am planning on sticking a tet cassette in between these genes such that the end of nifH and the beginning of nifD will be replaced by the cassette. My plan is to PCR a 1500 bp nifHD fragment, clone it into an integration vector for bacillus. Then cut in the middle of this insert and stick the tet cassette in there (pretty standard!)
I have a pair of conveniently located restriction sites in the sequence that correspond to the sites flanking the cassette in the source vector. However my problem is that by using these sites the cassette does not clone tight in the middle of this 1500 bp HD fragment. it leaves 884 bp on one side and 330 bp on the other. These two sequences flanking the cassette will be heavily relied upon for homology based recombination of the cassette into the chromosome, I am concerned whether this imbalance can have a negative bearing on recombination.

I don't know if this tidbit helps, but I do not see any difference in recombination efficiency in E.coli and S.pombe when the homology regions are of unequal length.

Hello all,

I have to disrupt a gene cluster (nifH-D) in a bacillus like bacterium of which very little sequence information is available. I am planning on sticking a tet cassette in between these genes such that the end of nifH and the beginning of nifD will be replaced by the cassette. My plan is to PCR a 1500 bp nifHD fragment, clone it into an integration vector for bacillus. Then cut in the middle of this insert and stick the tet cassette in there (pretty standard!)
I have a pair of conveniently located restriction sites in the sequence that correspond to the sites flanking the cassette in the source vector. However my problem is that by using these sites the cassette does not clone tight in the middle of this 1500 bp HD fragment. it leaves 884 bp on one side and 330 bp on the other. These two sequences flanking the cassette will be heavily relied upon for homology based recombination of the cassette into the chromosome, I am concerned whether this imbalance can have a negative bearing on recombination.
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Hi,
It largely depends on the organism you are dealing with and the size and function of the gene(s) you're trying to delete. In my experience with mycobacteria it is difficult to perform a gene disruption with flanks of unequal lengths. A difference of upto 200 to 250 bp is permissible but I havn't tried gene disruption with a difference greater than 250. For now, you can try the unequal flanks that you have and see whether it works. Good luck!

CD