expression of promoter T7 - (Jun/19/2011 )

hello everyone!

I have a tissue to discuss with everybody and look forward to sharing the help of you all. Currently, I am conducting a study of the design expression vector of the genes encoding the enzyme lycopene biosynthesis in E. coli. Here the vector expression that I use is the pRSET-A and alpha strains DH5 alpha as the host cell. The pRSET-A, the gene will be attached to the system controlled by the T7 promoter, this promoter requires the enzyme T7 RNA polymerase transcription to occur. That in DH5 alpha strain without this enzyme. However, we obtained the expression of the lycopene colonies. So why this happens?

Please anyone help!

hello!

Have a "tissue"? hahaha....sorry just joking around

First did you confirm that DH5 alpha is lacking the genes encoding the enzyme lycopene biosynthesis?. I was guessing that DH5 alpha is containing that gene...so when you check the expression of course you will get the protein...It is very important to get compatibility between host-plasmid..If you want to express some gene in e.coli mke sure there's such gene in that host...If you not sure either DH5alpha contain that gene, first try to culture the cell

1.check the expression of DH5alpha without plasmid...
2.check the expression of DH5 alpha with pRSET-A plasmid (No IPTG added)
3.Make an expression of DH5 alpha with PRSER-A plasmid and add some IPTG conc

Ok what we expect from this:
if you get the band for all lanes, same expression level mean..the wild type of DH5alpha contain the gene that encoding the enzyme,
if you get the (3.) get thicker expression mean the promoter T7 really work, as we all know the T7 can get induced by presence of IPTG...if you see there's no diff between (2) and (3)..mean that the T7 polymerase is not the issue here (not even a tissue...lol joking only)...it was come from the host...the host capable to express gene(because gene is there in the genome with original promoter)...

so if that case, try to use host another E.coli that losing that gene...Did i tell u clearly? correct host will show you the different before and after you induce with IPTG

This is what was cross in my mind

I don't think E. coli makes lycopene natively, but I could be wrong. More likely, you have transcription happening from a promoter you haven't identified. This could be a cryptic promoter in your operon, or read-through transcription from a promoter on the plasmid. You might try putting your operon in the reverse orientation in your plasmid. Or changing plasmids to one that reduces this read-through (Lucigen makes some, I believe).

I don't understand... how can putting the operon in reverse orientation in the plasmid can help?

p/s: hmn... lucigen... lately I heard people talked about it quite frequently... a new company?

If you have transcription activity arising from the plasmid, then the direction of the insert can determine if the transcript is in the sense or anti-sense direction. Lucigen is a U. Wisconsin Madison spinout that has been around for about 5-7 years, I think. They make a series of interesting cloning vectors.

Hi phage434, Many thanks for the enlightnement.

Cheers,
Adrian

Hi, Everyone
I have the same problem. I cloned a Y.pestis gene into pET1.2 cloning vector. This vector contains T7 promoter as well. I just checked the protein expression in DH5a harbouring this pJET1.2+Y.Pestis gene and I found very high level of protein expression without any IPTG induction. I was not expecting the desired Y.pestis protein expression from this construct as E.coli DH5a cells dont have T7 RNA polymerase to start transcription from T7 promoter. The gene is in right orientation, confirmed by restriction digestion and sequencing. Could anyone help me please why I am getting expression in DH5a cells without any IPTG induction as well.
Many Thanks