sucide plasmid directed mutagenesis - sucide plasmid directed mutagenesis in pseudomonas (Oct/22/2004 )

Dear Friends

What is sucide plasmid directed mutagenesis.Can you suggest a protocol for that.

Is the Tn10 derivative in plasmid vehicle are Sucide Vector.In some plasmid vehicle having trasposon there id written CamR and Plac or URA3 or Lacz.What is mean.Please suddest aprotocol how can I use them for mutagenesis.

Can I use Gene pluser or CaCl2 trasformation to introduce that trasposon containing plasmid in Pseudomonas.What will be marker to select the trasformatants.

Thanking you in advance
Regards
Manoj Kumar

A suicide plasmid is simply a plasmid which cannot replicate in the transformed cell. They would be grown up in another cell line (presumably E. coli, in this case). When transformed into Pseudomonas, the plasmid cannot replicate. The transposon carried by the suicide plasmid will (rarely) insert into the chromosome of the target organism. The transposon carries an antibiotic selection cassette. You need to find out it what antibiotic it carries. After transformation and growth of your target organism, you select with that antibiotic. Since it is a suicide plasmid, only cells that have undergone a transposon insertion event (into a non-lethal location) will grow in the presence of the antibiotic.

You might want also to look at the transpososome techniques being marketed by Epicentre.

I'd suggest electroporation as the easiest way of transformation.