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Replacement of Promoter - To replace araBAD promoter to T7 promoter (Dec/09/2009 )

Hi there,

I had recently cloned a gene into pBAD vector. For some reason, I would like to change the araBAD promoter to a T7 promoter after I had clone in the gene.

Is there anybody you know who had done this before?

Thanks.

Adrian.

-adrian kohsf-

Hey, i dunno 'bout nyone who has done this..........but I don't see any problem in this.......u just have to take care of the reading frame, no???

-DRN-

HI DRN,
thanks for your fast reply. Perhaps I should ask: how to do it? using RE method or shall I use a suicide vector or any other way?
I was trying to search for protocols but couldn't find any....

:..(

Anyone can help?
Thanks.

-adrian kohsf-

Hi Adrian

RE method should work.....just remove the old one n clone the new one in....

-DRN-

i think it should be much easier to cut the gene by RE and clone it into a novel vector used for T7 expression (like pET vectors from Novagen).

If you really want to exchange the ara promoter, cut it with appropriate REs, gel purify your vector backbone and ligate a synthetic T7 promoter that you make from two overlapping oligos (with the right overhangs for your REs) by annealing them (heat to 99C and let them cool in a controlled environment in a thermocycler) ...but for this task you should be somehow experienced.

If you don't have clue how to do it this way i would suggest to clone your gene in a pET vector, because it will be much easier and less labor-intense.

Regards,
p

-pDNA-

adrian kohsf on Dec 9 2009, 06:39 AM said:

Hi there,

I had recently cloned a gene into pBAD vector. For some reason, I would like to change the araBAD promoter to a T7 promoter after I had clone in the gene.

Is there anybody you know who had done this before?

Thanks.

Adrian.


RE works fine and is quite simple. You do not even have to remove the ara promoter...just clone the t7 into it if it's easier with respect to RE choice. I analyze predicted endogenous promoter elements by cloning them upstream of GFP and all I do is disrupt the plasmids original promoter.

As a control...also construct your new t7 promoterBAD vector with the promoter oriented in the wrong direction to drive expression of your gene. if successful, you only get expression from the new promoter construct in the correct orientation.

-eldon-

Thanks for all of your advices. I will do my best.

-adrian kohsf-