T7 and DH5 alpha
Posted 04 February 2010 - 02:57 AM
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?
Posted 04 February 2010 - 05:14 AM
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.
Posted 04 February 2010 - 01:37 PM
I have tried researching the subject now, but all it does is getting me more confused. I found this in some Invitrogen information document:
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?
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.
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?
Posted 04 February 2010 - 05:14 PM
You might find this E. coli genotype page useful: