E.Coli Plasmid<--->Chromosome Insertion/Recovery?

[Brad Nicholson]

In article <32F75333.4CBD at igmors.u-psud.fr>, Mark Blight
<blight at igmors.u-psud.fr> wrote:

> Hello,
> 
> I need to be able to transfer a gene onto the E.coli chromosome, study 
> single-copy expression and mutations and then recover the gene to a plasmid 
> again.
>snip< 
> I should perhaps try lambda phage and isolate a lysogen.
> 
> Can anyone suggest a technique to use that I haven't come across yet?
> 
> Thanks for any help.
> 
> Mark

Hi Mark,

You may know about this system already but here it is;  Simons, Houman &
Kleckner.  1987. Improved single and multicopy lac-based cloning vectors
for protein and operon fusions. Gene(53) p.85-96.  The system works really
well for E. coli.  You can clone your promoter into BamHI or EcoRI sites,
move it onto the chromosome with a lambda phage and recover onto a
plasmid.

Another idea could be to hop the promoter-reporter construct onto the
chromosome with a mini-Tn5 system developed by Timis, but I'm not sure how
easy it would be to recover it.  The reference for that is Herrero, de
Lorenzo & Timmis. 1990. Transposon vectors containing non-antibiotic
resistance selection markers for cloning and stable chromosomal insertion
of foreign genes in gram-negative bacteria.  J. Bacteriol.
172(11)6557-6567.  Also, the next paper in the journal is also of
interest.

Good Luck,

Brad