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

Chris Boyd chrisb at hgu.mrc.ac.uk
Mon Feb 10 10:13:02 EST 1997


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 sing=
: le-copy =
: expression and mutations and then recover the gene to a plasmid again. The =
: genes that I am =
: working with are not present on the K12 chromosome (so homologous recombina=
: tion =
: with my DNA won't work). I have looked at the pOM40 (Raibaud et al., (1984)=
:  Gene, 29: =
: 231-241) plasmid for insertion into and recovery from the malP promoter; bu=
: t this plasmid =
: only provides an EcoRI site for cloning (a necessity due to its constructio=
: n and =
: functionality). Preferably, I would like a MCS for cloning purposes. I have=
:  considered that =
: I should perhaps try lambda phage and isolate a lysogen.
: Can anyone suggest a technique to use that I haven't come across yet?

WARNING: this is a shameless self-advertisement...

You might want to look at the following paper:

  author =       "A. C. Boyd and D. J. Sherratt",
  title =        "{The pCLIP plasmids: versatile cloning vectors based
                 on the bacteriophage $\lambda$ origin of replication}",
  journal =      "Gene",   
  volume =       "153",
  pages =        "57--62",
  year =         "1995",

These vectors have the potential to exist as multicopy plasmids in the
normal way but can be made to integrate by homologous recombination
into prophages in appropriate strains. They also have MCSs available.

You can view a PostScript version of the manuscript on my web page (see
sig for URL).

Best wishes,
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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