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T7 and DH5 alpha


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#1 Axel

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Posted 04 February 2010 - 02:57 AM

Hello!
I'm doing my masters thesis and I'm trying to clear some things out so that I understand them fully. Now I'm investigating the T7 expression system. If I understand things right, the T7 RNA polymerase is usually incorporated into the chromosomal genome under control of a lac operator starting transcription with the addition of IPTG. Then, the polymerase finds the T7 promotor on the plasmid and starts transcribe mRNA from this site.

However, I read somewhere that my bacterial strain, DH5 alpha, does not carry its own T7 RNA polymerase gene, and that this should be present on the plasmid and need to be transformed. Is this true? I'm suspecting it's not, because I can't see that my plasmids (pTZ19R and pSE420) contains any such thing. Could you help me clear things out?

#2 phage434

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Posted 04 February 2010 - 05:14 AM

What you read is correct. Most T7 expression is done in strains of bacteria that have been lysogenized with the DE3 phage, carrying the T7 RNA polymerase under control of the lac promoter. DH5a does not have this. Usually strains having this modification are indicated in their name, such as BL21 (DE3). The reason DH5a does not typically carry this modification is that it is poor for protein expression, since it still has the lon protease, which tends to degrade expressed proteins. The BL21 strain is knocked out for this and other proteases.

You could certainly carry the lac promoter and T7 RNA polymerase gene on a plasmid (either the same one or a co-transforming one) in DH5a. Few people do this, because they care about the protein expression resulting.

#3 Axel

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Posted 04 February 2010 - 01:37 PM

Thank you for your answer! I was hoping I got things wrong so they would make more sense though.. I went back to my original source, and they clone my gene into a pTZ19R expression vector and transform into DH5a for expression. If DH5a is a poor gene expression strain, why would anyone want to do that? And how does expression take place if there is no T7 RNA polymerase?

I have tried researching the subject now, but all it does is getting me more confused. I found this in some Invitrogen information document:

Important: DH5α E. coli does not require IPTG to induce expression from the lac promoter even
though the strain expresses the Lac repressor. The copy number of most plasmids exceeds
the repressor number in the cells. If you are concerned about obtaining maximal levels of
expression, add IPTG to a final concentration of 1 mM.

My original source does not mention IPTG at all, and I assumed it was because they didn't want to give all information around the production of the protein, but maybe they just don't use it?

Also, I have noticed that I get clearer results on my SDS-PAGE while using a method involving PMSF. My supervisors opinion is that it is not needed, but I guess it would block the lon protease and have some sort of effect? I'll probably do some more test on that, just for personal amusement..

Ok, I'm just babbling away with my questions, but the first ones are the most important. Where's the polymerase?

#4 phage434

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Posted 04 February 2010 - 05:14 PM

The pTZ19R plasmid has BOTH a lac promoter and a T7 promoter upstream of its multiple cloning site. In a LacI- strain, this will express from the lac promoter, having nothing to do with the T7 promoter. The T7 promoter is there to allow expression in a BL21 (DE3) strain. If I cared about the protein, I would subclone into BL21(DE3) and induce with IPTG, then (as you are doing) purify in the presence of PMSF, although that is less important in BL21 strains.

You might find this E. coli genotype page useful:
http://openwetware.o..._coli_genotypes




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