How does triparental mating work?

Lim Jet limjet at pl.jaring.my
Mon Sep 21 05:44:05 EST 1998


Hello,
	My name is Lim and I am a student in Ngee Ann Polytechnic, Singapore. I
am currently working on a project that involves the transformation of
Agrobacterium tumefaciens via triparental mating.My donor cell is XL1
Blue and my helper strain is E.coli DH5 alpha. I have some queries on
this method of transformation and was wondering if you could help me.
The following are the questions that I would like to ask:

1) I noticed that most of the papers published involved using a
conjugative plasmid    pRK2013 in the helper strain. 
   What is this plasmid for? 
   Is this plasmid really necessary if we want to transform
Agrobacterium with this    method? 
   Is pRK2013 a  plasmid that codes for a conjugative pili that will be
specific for    Agrobacterium?
   Can any other conjugative plasmid be used? For eg. can a F plasmid
that is usually      found in E.coli be used instead?

2)What is the mechanism behind triparental mating? I mean how does the
Agrobacterium eventually get transformed with our plasmid of interest
from the donor cell in the presence of helper cells? My donor and helper
cells are E.coli of diifrent strains.

	I hope to receive a reply from you soon. Your assistance will be
greatly appreciated. Thank you.

Your Sincerely,
Lim
limjet at pl.jaring.my



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