Suicide vector - how to do (Oct/24/2005 )
Hi, I am new at molecular biology and would like to ask you all.
A few weeks ago I have ligated foreign gene into PblueScript SK2 +
Then Now, I want to integrate the gene into other bacteria's chromosome
I have heard about "Suicide vector" elsewhere, but wondering how to get the plasmid and how to start my experiment. One of my frineds told me that if I have an electroporation, I can just use any kind of plasmid derived from PUC19 or 18.
Is there anyone who is familiar with this type of experiment?
i've started a topic a month ago in a hurry for helping a colleague that had an exam (how do you say "concours" in english??....) So excuse me for the lack of informations but that may help you.
you can find it here:
thanks Fred, so it seems like you also integrated gene into chromosome.
How did you do? Could you tell me what plasmid you used and how you transfer the plasmid into bacteria. Was it, well, no problem that the plasmid gets suicide and integrated in chromosome?
Please let me know,
well to be honnest, i did not get all your topic at first reading.
For intergrating gene into the chromosome, i think that suicide gene is not the better way.
i supposed you need a gene that when expressed killed the bacteria.
But you mean by suicide vector a vector that can't express itself and replicates or sthg that enables destruction of it?
Can't it simply possible to produce the plasmid you ant in big quantity, and digest it in order to remove bacterial replication origin, make blunt ends and religate. Transform bacterias and apply selection pressure to select bacterias that had integrated the defective plasmid?...
By far the easiest chromosomal modification system in coli is found in the Lambda RED work of Don Court. Specifically, his DY329 strain of coli can be edited with a simple heat shock and transformation with linear DNA containing 35-50 bp of homology to the desired insertion site on each end of the insert.