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help with p16slux tagging protocol

p16slux luminescent

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#1 howardjli

howardjli

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Posted 25 August 2012 - 03:07 PM

Hi all,

I'm trying to tag a strain of Salmonella using the p16slux method (http://www.ncbi.nlm....les/PMC2074938/)

Basically, p16slux is a plasmid with temperature sensitive replication that will site-specifically integrate both its luxCDABE operon and erythromycin resistance cassette into the 16s locus of gram-negative bacteria genomes (though integration happens at a very low frequency). This results in bacteria that are constitutively luminescent.

The protocol is straight forward: transform p16slux into target bacteria, incubate transformants at 30 C with erythromycin. Grow liquid cultures from single colonies at 30C with erythromycin. Dilute liquid cultures and grow at 42 C with erythromycin . Dilute culture and spread on an LB agar plate with erythromycin, 42 C. Growing up cells at 42 C prevents plasmid replication and only allows cells that have undergone the integration to survive.

Problems I've encountered
  • Cells growing up at 42 C are resistant to erythryomycin, but nonluminescent or only weekly luminescent.
  • Cells testing positive for integration (confirmed via PCR) are also nonluminescent.
Has anyone else tried this protocol? Please advise!

Howard




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