Construction of E.coli strains
tsu01135 at KORYU.STATCI.GO.JP
Mon Jun 30 05:11:52 EST 1997
On June 27, Martin Messerle wrote:
>we are working with an E.coli strain which has certain useful
>properties. We would like to delete the recBC functions of this
>strain. Can anybody tell me how to do it and how complicated is the
>procedure? I have been told that it can be done by P1 transduction?
Ihnen wurde richtig gesagt. The manipulations are not difficult, but
experience in bacterial genetics would be helpful. The most
complicated part is simply finding the appropriate strains to use.
Basically you want to find an E. coli strain that has a mutation in
recB or recC, and use P1 to transduce that mutation into your strain.
The catch is that you need to be able to select for something.
Generally, a nearby Tn10 insertion is used for selection. If you can't
find a suitable strain, then you first have to transduce a suitable
Tn10 into the mutant strain before transducing the recB or recC
mutation into your strain of interest. The trick here is to be neither
too close nor too far away with the Tn10 insertion. You'll have to
screen the resistant colonies (with the transduced Tn10) to find the
ones that are still mutant for recB or C (i.e. sensitivity to methyl
methane sulfonate). If the Tn10 insertion is too close, these will
be rare. If it is too far away, this part will proceed fine, but you'll
have trouble using it to cotransduce the mutation into your ultimate
Unfortunately, I don't know of any web sites describing P1
transduction, but there is a wonderful two volume set of books that
should be available in your library, or even in your personal
collection. Cold Spring Harbor Press has published a much needed
update to Jeffrey Miller's 1972 Experiments in Molecular Genetics.
The updated 1992 publication consists of two volumes, a manual and
a handbook. It's by the same author, and is entitled "A Short Course
in Bacterial Genetics: A Laboratory Manual and Handbook for
Escherichia coli and Related Bacteria. You can find info at
As for finding the bacterial strains you need, visit
The E. coli Index at:
from there, click databases, and from there click CGSC: E. coli Genetic
Stock Center. I found it a little confusing to search their database,
but a little perseverence pays off.
By the way, regarding selection for loss of tetracycline resistance,
if your resistance is due to insertion of a Tn element into a selectable
gene, you may find it more effective to select for recovery of that
gene function on appropriately supplemented minimal medium plates.
In my experience, clean excision of some Tn elements is rare, and you
may have to plate out as many as 10*9 to 10*10 bacteria to get the
clean excision you want.
Robert F. Whittier, Ph.D.
Mitsui Plant Biotechnology Research Institute
TCI D-21, Sengen 2-1-6
305 Tsukuba, Japan
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