Will this sequence work as promotor?

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Dorif

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May 29, 2015, 7:09:39 PM5/29/15
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Sequence: ATTGTGAGCGGATAACAAAAACTTGACTAAAGATTCCTTTAGTAGATAATTGTGAGCGGATAACAATTC

It is based on p22 Salmonella phage promotor with replacement of OR1 and OR3 repressor binding sites by lactose operon repressor binding sites from Escherichia coli. How probable is what this construction will work?

Mega [Andreas Stuermer]

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May 30, 2015, 7:01:58 AM5/30/15
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 Extremely hard to say... I just know from T7 if you space lacO (or was it tetO) more than 5 bp away it doesn't work anymore because the DNA helix. The repressor will bind the operator but in a different angle... Have you made sure that the core promoter will be in the same position relative to the operator? Isn't it possible to take the native coli one?

Александр Дориф

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May 30, 2015, 7:59:13 AM5/30/15
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Yes, it remains in exactly the same position relative to the operator. I think it is preferable to use Salmonella phage promotor for expression in Salmonella, doesn't it?

2015-05-30 14:01 GMT+03:00 Mega [Andreas Stuermer] <masters...@gmail.com>:
 Extremely hard to say... I just know from T7 if you space lacO (or was it tetO) more than 5 bp away it doesn't work anymore because the DNA helix. The repressor will bind the operator but in a different angle... Have you made sure that the core promoter will be in the same position relative to the operator? Isn't it possible to take the native coli one?

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Mega [Andreas Stuermer]

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May 30, 2015, 10:02:00 AM5/30/15
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E coli and Salmonella are more or less related. So I guess the coli promoter woud likely work without modification. Does Salmonella have a lac repressor?

Michael Tellier

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May 30, 2015, 10:42:34 AM5/30/15
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Interestingly, most Salmonella genomes have lost the lac operon, which seems to hinder its pathogenicity by down-regulating the genes involved in the flagelles. So if you put your construct in Salmonella, your gene will be always expressed since LacI is likely to be absent. You might need to add the LacI gene on your construct to express the repressor. 

About Salmonella and the absence of the lac operon:

Dorif

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May 30, 2015, 10:44:16 AM5/30/15
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No. No one of Salmonella species has possibility to use lactose, so they don't have any parts of lactose operon. LacI should be inserted simultaneously with LacY and LacZ. Also, I've analysed Salmonella 16s rRNA sequencies on NCBI and there are differencies in RBS of E. coli and Salmonella(but yes, they are VERY similar). But, when I'll obtain "building blocks" with E.coli promoters and RBS I'll try to use them as well. And if I'll obtain not very good results - I'll try to use this.

Dorif

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May 30, 2015, 12:55:07 PM5/30/15
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Interesting info. Thanks a lot! Are known functioning modifications of LacI, LacZ, LacY? What if prducts of these genes would be LVA marked for faster degradation?
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