How to circumvent DNA restriction in a bacterial host?

M. Alexeyev malexeyev at biost1.thi.tmc.edu
Tue Nov 21 11:15:32 EST 1995


In article <48s9u1$rvt at nntp.ucs.ubc.ca>, bina at unixg.ubc.ca (Jim) wrote:

> Hi,
> 
> I am trying to do gene replacement in a gm(-) bacteria that contains an 
> uncharacterized restriction/modification system(s), however, I am not able to 
> get recombinant transformants via electroporation or CaCL2 methods.  I assume 
> the problem is DNA restriction since I had a tough time cloning DNA from this 
> organism.  I tried using several different R/M E. coli strains for
preperation 
> of DNA without success.  Does anyone have any suggestions or ideas on
how I can 
> get arround this problem?
> 
> Thanks Jim.

Dear Jim:

It is difficult to suggest you anything without knowing particularities.
What bacteria are you working with? Are you able to introduce homologous
DNA from a related bacterium?   Do you have any evidence that either CaCl2
procedure or electroporation protocol that you used are efficient enough
to both introduce DNA AND achieve homologous integration of that DNA into
the chromosome of the recipient (personally, I would doubt CaCl2 procedure
very much)? Are you sure that selective marker is being expressed in a
recipient (there are examples when it doesn't)? Any clues on type of
restriction/modification system that may be present in the strain?

 Without knowing this, I may only suggest to:

1. Try conjugation. It normally works for insertion of homologous DNA into
the chromosome of R/M+ cyanobacteria (type II R-M systems). You may also
try introducing the broad-host-range plasmid (like RP4 or RSF1010) into
your strain by conjugation. RP4 codes for Km, Ap and Tc resistances and
RSF1010 codes for Sm-res (if I am not mistaken), so you will be able to
test several selection markers. Since there is no warranty that either RP4
or RSF1010 will replicate in  a recipient bacteria (although, there is a
decent probability), you may try introducing transposons on a suicide
plasmid simultaneously.

2. Try to get some idea of the nature of R-M system present. Detection of
the typeII R-M systems can be as simple as sonication of the cell
suspension in tris-NaCl-2-ME solution and incubating the cell lysate with
substrate DNA (lambda, AD2, your favorite plasmid). 

Good luck,
M. Alexeyev



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