I am planning to make mutant in Francisella novicida using suicidal vector. For designing primer set i need clear conception about principle of action of suicidal vectors. Can anybody send me the link/site for easy understand?
The principle is easy. You have a vector with a selectable marker that has an origin that maintains it in one organism (usually E.coli) but is inoperative in the other.
If you transduce that vector from the competent host (e.g. E coli) into Francisella novicida (where the origin doesn't work), the plasmid is rapidly lost unless it integrates. You can select for integrants with the selectable marker. The usual stunt you are trying to pull is homologous recombination into a specific site. To achieve that, you put a fragment of the Francisella novicida target sequence into your vector so that homologous recombination between the vector and the Francisella novicida genome is promoted. Amongst the integrants will be a subpopulation having the vector recombined into your chosen target site.
Thank you Sir for your nice explanation. Now I am using pFRSU plasmid. Successful rate of homologous recombination is very very low.
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