Construction of E.coli strains

[R.F.Whittier]

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 
target strain.

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 

http://www.cshl.org/books

As for finding the bacterial strains you need, visit 
The E. coli Index at: 

http://sun1.bham.ac.uk/bcm4ght6/

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.

--Bob

Robert F. Whittier, Ph.D.
Senior Researcher
Mitsui Plant Biotechnology Research Institute
TCI D-21, Sengen 2-1-6
305 Tsukuba, Japan