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How to find promoter sequence ...

...of Lactobacillus LDH?

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3 replies to this topic

#1 rmbio

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Posted 12 December 2014 - 03:19 AM

Hi all

 

I wish to use lactate dehydrogenase promoter from Lactobacillus acidophillus for heterologous expression of some genes under it in the same species. I saw few papers like this and this in which they have used the same promoter and have given the primer sequence to isolate it. But neither any reference has been given on the source of this primer sequence nor the promoter sequence itself been reported. Since I want to clone the promoter (from different strain), I wish to know its sequence (and size) so that I can choose appropriate restriction enzymes for cloning.
Lactobacillus acidophillus genome is already known and is available at here and here
How can I go about finding out the LDH promoter sequences so that I can design the primers with appropriate RE site and go ahead with cloning?

Thanking you!
 



#2 phage434

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Posted 12 December 2014 - 07:06 AM

Bacterial promoters are usually quite close to the gene. First, identify the N terminal protein codon (usually an ATG). Look upstream for the RBS (usually a sequence containing AGGA) about 8 bp upstream. The promoter is usually upstream of that, from 6-several hundred bp. For the low GC organisms such as lactococcus, the promoter usually has a -10 region of the form TAAAAT (critical spacing of the two T's). There is often a -16 region, TGN just upstream of the -10 region. These organisms often omit the -35 region, which (if present) would be of the form TTGACA spaced 17-18 bp upstream of the -10 region. This -35 region is much less conserved, and may be missing entirely.

I would suggest that you locate the primers in the literature in the strain they took it from. Likely the new strain will have very similar structure. You may even be able to use the same primers.



#3 phage434

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Posted 12 December 2014 - 07:09 AM

Also, have you considered simply using the reported promoter? It almost certainly would work in your species.



#4 rmbio

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Posted 14 December 2014 - 09:41 PM

Thank you Phage434. It was really a helpful reply. I have found the primer region in the upstream of LDH gene and would be using the same region.

 

Thanks






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