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How to select promoter for resistance gene in linear/naked DNA "knockout"

Microbiology Knockout mutant Promoter selection Homologous-recombinatio

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#1 Chris22

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Posted 30 March 2015 - 10:43 AM

Hello,

 

Short version:

How do you pick a constitutively expressed promoter from a bacterial genome (to place upstream of an antibioticr gene in a knockout cassette) when you have the genome sequence? 

 

 

Long version:

I would like to design a linear (naked) DNA cassette to create a knock-out mutant by homologous recombination in a Bacteroides species.

 

I know that approx 500bp upstream and downstream of the target gene must be fused to a suitable antibiotic resistant cassette (Tet or Ery) but I am concerned that the current promoter region may not function in my bacterial species.

 

I would like to fuse a promoter from my bacteria to regulate expression of this resistance gene instead. The genome is sequenced but no genetic system exists for this bacteria.

 

Would anyone know if there are rules for picking a promoter for this purpose?

 

Does it have to be a housekeeping gene promoter that is always "on"?

 

Are there standard ones normally used, 16S/HSP/etc promoters?

 

Is it just trial and error with different ones?

 

Thanks in advance

Chris


Edited by Chris22, 30 March 2015 - 10:43 AM.


#2 phage434

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Posted 30 March 2015 - 05:20 PM

Sigma 70 promoters are quite functional in many bacterial species. Are you working with a gram negative or positive organism? What GC content do you have?

Common strong promoters are the ones for EF-TU, for 16S rRNA.



#3 Chris22

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Posted 31 March 2015 - 07:01 AM

Thank you for replying phage434!

 

I'm working with gram negatives, 2 different genus actually, one Porphyromonas and a Prevotella species.

 

The GC contents are 45% and 41% respectively,

 

That is a great help actually, I had not considered sigma factor promoters.

 

If someone has actually done this and fused a promoter from a sigma factor/16S/EF-TU to an antibiotic resistance cassette I would really like to know if it was successful?







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