Transposon Mutagenesis HELP PLEASE

[Mikhail Alexeyev]

In article <mjones-1907960945400001 at virol11.rpms.ac.uk>, mjones at rpms.ac.uk
(Mick Jones) wrote:

> Hi everybody
> 
> I wish to do the following type of experiment, and was wondering if it had
> been done before by anybody, or even if it will be a waste of time (i.e.
> it is a stupid idea).
> 
 
> Problems I can foresee are;
> 
> 1.  You may get multiple plasmids transfected and therefore have more than
> one integrationn event.
> 

This hardly can be a problem. At least in Tn10 case it has been shown that
donor molecule is destroyed after transposition. The frequency of
transposition rearly exceeds 10^-3. It is unlikely to get 1000 DNA
molecules into a single cell by electroporation or transformation. If
cannnot get more 10^3 DNA molecules into a single cell, you will not get
multiple inserts, unless donor molecule can replicate in recipient strain.

> 2.  The expressed transpoase may not work in the target cell.

If it works with vectors transferred by conjugation, it should work with
vectors introduced by electroporation or transformation. I know of at
least two people who used electroporation successfully to introduce
vectors similar to ones described by Lorenzo and Timmis  (Gene v160 p59,
1995) in E.coli

> Am I missing something?  Will this idea work?  Anybody tried it?
> 
> Please email me directly as well as replying to the news group.
> 
> Thanks in advance
> 
> Mick
> 
 
I think the problem with alternative ways to deliver suicide plasmids is
that they are not as efficient as conjugation (up to 100 % of recipient
cells can receive a plasmid from donor in some cases). Therefore, in
chemical transformation or electroporation experiments you will have
numbers working against you, i.e. if transposition frequency in your bug
is 10^-4 per DNA molecule and electroporation delivers an average of 10
DNA molecules per cell, you must be able to achieve electroporation
efficiency of at least 10^6 transformants/ug DNA in a typical
electroporation using 100 ng of plasmid DNA in order to get 100
"transposants". 

Of course, if conjugation doesn't work...

M.A.