E.coli "gene knockout": help
Giuseppe Borsani
E.coli genome when no selectable phenotype is available for the gene to
inactivate?
Probably it's something obvious for people with experience in bacterial
genetics. References and protocols are welcome.
Thanks
/ }
{ / Giuseppe Borsani
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Robert Solomon (Bioc)
>Does anybody have any suggestion on how to create a "gene knockout" in the
>E.coli genome when no selectable phenotype is available for the gene to
>inactivate?
>Probably it's something obvious for people with experience in bacterial
>genetics. References and protocols are welcome.
References for gene knockouts in coli..
Gutterson, NI & Koshland, DE Jr, Proc. Natl. Acad. Sci. USA 80, 4894 - 8 (1983)
Gay NJ, J. Bacteriol. 158, 820 - 5 (1984)
Basically, these involve a temperature sensitive polA mutant strain, wherein
the plasmid carrying the mutated gene cannot replicate extrachromosomally at
the restrictive temperature - thus, growth on antibiotic at restrictive temp
indicates a recombination event (hopefully a homologous recombination event).
This leaves you with two chromosomal copies of the gene, separated by the
plasmid DNA. Selection of segregants (loss of plasmid ) should leave you
with a mix of wt and mutant bugs. How you select between them is up to
you - if you have no screen for function then I suspect it's up to PCR
and sequencing to rescue you - I'm having no joy PCR'ing from either
colony scrapings or chromosomal DNA preps, but I'm assured that these are
possible. P1 transduction is then used to take the mutation into the
background strain of your choice. Have fun.
Good luck
Rob Solomon
Richard_Heath
> bor...@dibit.hsr.it (Giuseppe Borsani) writes:
>
>>Does anybody have any suggestion on how to create a "gene knockout" in the
>>E.coli genome when no selectable phenotype is available for the gene to
>>inactivate?
>>Probably it's something obvious for people with experience in bacterial
>>genetics. References and protocols are welcome.
>
> References for gene knockouts in coli..
>
> Gutterson, NI & Koshland, DE Jr, Proc. Natl. Acad. Sci. USA 80, 4894 - 8 (1983)
>
> Gay NJ, J. Bacteriol. 158, 820 - 5 (1984)
>
> Basically, these involve a temperature sensitive polA mutant strain, wherein
> the plasmid carrying the mutated gene cannot replicate extrachromosomally at
> the restrictive temperature - thus, growth on antibiotic at restrictive temp
> indicates a recombination event (hopefully a homologous recombination event).
> This leaves you with two chromosomal copies of the gene, separated by the
> plasmid DNA.
Another way to knock the gene out is to put an antibiotic resistance marker
into your cloned gene in a plasmid, then transform with linear DNA obtained
from this new plasmid. Homologous recombination should insert the
gene::antibiotic marker construct into the chromosome in place of the original
gene. This method has been used successfuly in our lab recently - don't have
any refs to hand right now though.
>Selection of segregants (loss of plasmid ) should leave you
> with a mix of wt and mutant bugs. How you select between them is up to
> you - if you have no screen for function then I suspect it's up to PCR
> and sequencing to rescue you - I'm having no joy PCR'ing from either
> colony scrapings or chromosomal DNA preps, but I'm assured that these are
> possible.
Yes, PCR does work, and is the best (only?) way to go if you don't have a
phenotypic/enzymatic assay (even if you do have an assay, it is still the way
to go!) (genomic DNA preps from selected colonies)
> P1 transduction is then used to take the mutation into the
> background strain of your choice. Have fun.
Correct.
>
> Good luck
Seconded.
>
> Rob Solomon
Richard Heath
Chris Yost
> > with a mix of wt and mutant bugs. How you select between them is up to
> Yes, PCR does work, and is the best (only?) way to go if you don't have a
> phenotypic/enzymatic assay (even if you do have an assay, it is still the way
> to go!) (genomic DNA preps from selected colonies)
>
> >
> > Good luck
>
> Seconded.
> >
> > Rob Solomon
>
> Richard Heath
There is a way to directly select for gene replacement. In other words
directly select for a double recombination event (ie no wt bugs). The
strategy is described in Quandt and Hynes, 1993. Gene 127:15-21.
Basically it involves placement of the sacB gene in your suicide deliver
vector. In the presence of sucrose, expression of the sacB gene is lethal
in gram negative bacteria. Therefore after you have placed you suicide
vector construct in your bug, selection with your antibiotic resistance
marker on media containing 5% sucrose should yield bugs that have
undergone a double recombinational event (ie they did not incorporate sacB
into their genome). To verify proper insertional inactivation of your
gene a Southern blot using your gene as a probe should show an increase in
size corresponding to the insertion of the antibiotic resistance gene.
I hope my description is clear and that this info helps.
Chris Yost
cy...@acs.ucalgary.ca
Rafael Maldonado
>
> Another way to knock the gene out is to put an antibiotic resistance marker
> into your cloned gene in a plasmid, then transform with linear DNA obtained
> from this new plasmid. Homologous recombination should insert the
> gene::antibiotic marker construct into the chromosome in place of the original
> gene. This method has been used successfuly in our lab recently - don't have
> any refs to hand right now though.
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
__________________________________________________________________________
Rafael Maldonado "You gave to a boy the job
Department of Human Genetics of a man. He's dead."
University of Utah
Salt Lake City, Utah 84112. USA. John Wayne
Raf...@genetics.med.utah.edu
Raf...@corona.med.utah.edu "No por mucho madrugar, amanece
Tlf: 801-581-4429 mas temprano"
Fax: 801-585-3910
Saber popular
Brad Nicholson
Maldonado <rafael@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 |
br...@corona.med.utah.edu |
or: (801)-581-4365 | My opinions are solely my own.
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