E.Coli Plasmid<--->Chromosome Insertion/Recovery?
Brad_Nicholson at hlthsci.med.utah.edu
Mon Feb 10 05:51:59 EST 1997
In article <32F75333.4CBD at igmors.u-psud.fr>, Mark Blight
<blight at igmors.u-psud.fr> wrote:
> 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
> 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.
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
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
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