E.coli "gene knockout": help

Richard_Heath heath at mbcf.stjude.org
Fri Mar 10 12:39:36 EST 1995


In article <3jf20p$6et at lyra.csx.cam.ac.uk>, rgs at mole.bio.cam.ac.uk (Robert Solomon (Bioc)) writes:
> borsang at 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



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