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Will a Lac promoter sequence interrupt transcription?


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6 replies to this topic

#1 tihong10

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Posted 23 October 2013 - 12:45 AM

Hello,

 

I am planning to insert a promoter into a pDsRed-Express2 vector (see attachment) upstream of the lac regulatory features. I will be transfecting a non-lactose fermenting bacteria with this recombinant plasmid.

 

I wanted to ask if anyone sees a potential problem that the lac regulatory features might pose when the transcriptional machinery meets this sequence. I know that when transcription starts from within the promoter that the downstream features (rest of the promoter, operator, etc.) will be transcribed into the mRNA transcript, but I wasn't totally sure what would happen if the transcription starts from further upstream.

 

I want the mRNA transcript to be made all the way from the upstream promoter, past the lac promoter, and all the way to the end of the RFP gene so that translation of the RFP gene can be successfully carried out as a reporter of upstream promoter activity.

 

Does anyone see any potential early termination of transcription that would occur with this configuration before the desired transcript is formed? Maybe a potential problem with the lac reg features or a transcription termination sequence somewhere...

 

Thank you all in advance,

Cheers,

Ted

 

 

 

 

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#2 phage434

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Posted 23 October 2013 - 04:54 AM

This should work, although I suspect you will also see substantial transcription from the existing promoter. Why would you not simply replace the lac promoter with the one you want to study?



#3 tihong10

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Posted 23 October 2013 - 03:19 PM

Hi Phage,

 

I am transfecting Pseudomonas aeruginosa (14) which is a lactose negative bacteria so from my understanding they don't possess the machinery to initiate transcription of the lac promoter therefore I don't foresee any unwanted expression from the lac promoter. I have electroporated PA14 with the non-recombinant RFP plasmid and it doesn't express so I am quite certain I won't get unwanted signals.

 

The reason I don't cut out the promoter is because the RBS is downstream of the lac promoter and there is no restriction site available to keep the RBS but remove the lac promoter.

 

Cheers,

Ted



#4 phage434

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Posted 23 October 2013 - 03:28 PM

I believe you will see very significant transcription from this promoter. It is a standard sigma-70 promoter, with a lacI repression site. You won't see repression, but that simply will make it express all the time.

 

You can ALWAYS put a restriction site where you want it with PCR, so this a very poor reason not to make the construct you really should make.



#5 tihong10

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Posted 23 October 2013 - 07:00 PM

Could you elaborate on how I could place a restriction site into a target location in the plasmid? I would definitely like to go the route of cutting out the lac sites if I can...



#6 phage434

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Posted 24 October 2013 - 04:20 AM

Design primers to amplify the entire vector (starting at the RBS site of your protein of interest), and ending 5' of of the promoter you want to remove. Add restriction sites of your choice to the 5' end of each primer. Add 3-6 bases of junk DNA 5' of these sites. PCR with these primers, purify, digest, and ligate. You can add DpnI to the digestion enzyme mix, which will selectively cut the original plasmid template (it has A methylated GATC sites), which will reduce background. You should use as little template in the PCR reaction as possible to again reduce background.



#7 tihong10

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Posted 25 October 2013 - 01:01 AM

That's a great idea! Thanks for your help :)

 

-Ted






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