Please help

Ramanathan Vairamani rv21 at cornell.edu
Thu Sep 17 08:50:36 EST 1998


Hi Lim,

You cannot do a triparental mating without a helper like pRK2013 because
the binary plasmids (in general use) have only an OriT and not the genes
required for conjugal transfer.  Binary plasmids are based on Wide Host
Range replicons and hence can replicate in both E. coli and
Agrobacterium.  pRK2013 has both OriT and the genes required for
conjugation.  But pRK2013 is based on colE1 replicon and hence can
replicate in E. coli only.  

This is the sequence of events during triparental mating:

1.  pRK2013 moves into the E. coli strain containing your binary plasmid. 
Now this intermediate strain has both pRK2013 and your binary.  

2.  Proteins encoded by the mobilization genes of pRK2013 aid the conjugal
transfer of your binary into Agrobacterium. (I am simplifying things
here.  The matings can happen in a whole lot of combinations but they
don't matter here).

3.  Even if pRK2013 gets into Agrobacterium it won't be able to
replicate.  So only your binary survives in Agrobacterium


You have to make sure that you have two levels of selection for picking up
the correct transconjugants.  First level is to make sure you only get
Agrobacterium growing on the plates.  Second step is to make sure you
select Agrobacterium with the binary plasmid.  You can ensure growth of
only Agrobacterium (and not E. coli) by plating on AB minimal medium.  YOU
can select for the binary plasmid with the antibiotic resistance marker in
the vector backbone of the binary plasmid.  

If you are not sure about the protocol, e-mail me I can send you the
detailed protocol.

Hope this helps

Ram
 

In article <3601E0BC.431C at pl.jaring.my>, limjet at pl.jaring.my wrote:

> 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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