E.coli "gene knockout": help

Brad Nicholson brad at corona.med.utah.edu
Sun Mar 12 14:21:16 EST 1995


In article <Pine.SOL.3.91.950311223859.27232B-100000 at corona>, Rafael 
Maldonado <rafael at corona> wrote:

 >But you need use a rec(something)- strain. Others are not capable of 
 >linear DNA transfromation. I don't remember the rec gene, may be recB 
 >scb? Can someone pint us to this system?
 >
 >Rafa

 The mutation needed is recD.  A quick scan of ATCC on gopher gives the 
following strain.  (Reproduced without permission)
-------------------------------------------------
ATCC 47060  - Escherichia coli PMC103, host

J.P. Doherty. Constructed from N2680 by P1 transduction of
delta(mcrBC-hsdRMS-mrr) from E. coli ER1648. Genotype: mcrA
delta(mcrBC-hsdRMS-mrr)102 recD sbcC. This strain is being made
readily available to the scientific community with the proviso that
it not be used to form the basis of a product for commercial sale
or be included in any commercial product (personal communication).
Methylation tolerant, recombination deficient host for
bacteriophage lambda. Capable of maintaining sequences that form
secondary structure. Shows large plaque size (Gene (Amst.) 124:
29-35, 1993).
Growth Conditions: Medium 1065 37C
Shipped: freeze-dried.
Price Code: C
-------------------------------------------------
Alternatively, New England Biolabs has a recD strain available; ER1647 
(Order #401-N).  It is a derivative strain with a lot of other mutations.  
See 1995 NEB Catalog p.218.  P.S.  I don't work for NEB.
-------------------------------------------------
On the other hand Thomas Elliot has an E. coli strain with a 
recD::TN10d-Tet allele that could probably be mobilized to the background 
of choice with P1.
Reference: Thomas Elliot. 1992. J. Bact. 174:245-53. 
-------------------------------------------------
I have done knockouts with a recD background using electroporated linear 
DNA.  You need a ton of competent cells and a ton of absolutely linear DNA,
but I have been told that it is possible to use chemical competent cells 
and transformation.

Brad

===========================================================================
Brad Nicholson              |"If it worked the first time, it wouldn't be 
Department of Pathology     |	research."...Brad Nicholson
University of Utah          |	Live from behind the Zion Curtian.
Salt Lake City, UT 84132    | 
brad at corona.med.utah.edu    |
or: (801)-581-4365          | My opinions are solely my own.
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