Bacterial gene disruption problem - (Aug/06/2008 )

hi all,

I've been trying to make double mutant for a relatively new bacterium with few genetic manipulation system. Previously, my lab member can successfully knockout single gene in it by using pBK-CMV plasmid cloned with 1000bp flanking region of gene of interest, with a spectinomycin cassette integrated between the two flanking region. I'm now trying to make a double mutant, does it means that I must construct another plasmid in the same way and cloned in with another antibiotic marker gene?
In addition, I would like to know if there are any method to remove the integrated marker gene afterwards. I am afraid it may affect the genes downstream as the gene is located within an operon. There should be no suicide plasmid available for the bacterium.

Thanks in advance~

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If the spectinomycin cassette has not been removed than yes, you will have to use a second antibiotics marker.




Well you have two options
1- build your own suicide plasmid, with the appropriate strain background. To make the suicide system work you need a counterselectable marker.

2- use site specific recombinase such as Cre or phiC31 phage integrase to excise the marker. I would recommend using phiC31 as this integrase is unidirectional and thus you can use the phiC31 integrase system multiple times. Cre is reversible recombinase. Cre and phiC31 have been shown to work in a wide range of organism (eg S.pombe, S.cerevisea, human cell, chicken cell, mouse cell, drosophlia, e coli). So it should be reasonably safe, but you need to do a trial experiment to determine the the recombinase works in your organism. It is a rather high efficiency system.

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Thanks for your suggestion
actually i've considered of using lambda red recombinase system and had obtained a helper plasmid pKD46, but seems like this plasmid is not replicable in the bacterium i'm working on. So I'm thinking of making a modified version by integrating the origin of replication from plasmid of that bacterium. But if the temperature sensitive ori and rep should work in E.coli only, not really sure if the plasmid can be rescued after it's transformed in that bacterium.
Any advices?

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