pET-3b

[apricimo]

Is pET-3b used for expression?

 

 

If so how do you induce it? If you don't induce it how does it work? If it is not an expression plasmid what is it used for?

 

thanks

[Greippi]

-Yes, it is used for expression.

- Induce with IPTG or infection with lambda CE6

 

More info

Edited February 10, 2011 by Greippi

[apricimo]

-Yes, it is used for expression.

- Induce with IPTG or infection with lambda CE6

 

More info

 

But it doesn't have a lac operon... What does IPTG do?

[CharonY]

I assume you mean lac promoter (the whole operon is not needed and lacI is often provided by the host strain)? IPTG induces the lac operator, acting as a lactose-analog (essentially by binding to the lac repressor). The induction with IPTG is dependent on the host strain. The trick here is the use of the T7 promoter. In order to express the gene insert under control of the T7 promoter, the viral T7 RNA polymerase has to be present. This is done by having a host strain carrying it. If the T7 RNA polymerase is under control of a lac promoter, but is repressed by LacI, IPTG would induce T7 polymerase expression, which in turns results in expression of the insert in the plasmid.

Thus, the control of induction is not directly done by cis acting elements on the plasmid, but relies on a compatible host strain.

[apricimo]

I assume you mean lac promoter (the whole operon is not needed and lacI is often provided by the host strain)? IPTG induces the lac operator, acting as a lactose-analog (essentially by binding to the lac repressor). The induction with IPTG is dependent on the host strain. The trick here is the use of the T7 promoter. In order to express the gene insert under control of the T7 promoter, the viral T7 RNA polymerase has to be present. This is done by having a host strain carrying it. If the T7 RNA polymerase is under control of a lac promoter, but is repressed by LacI, IPTG would induce T7 polymerase expression, which in turns results in expression of the insert in the plasmid.

Thus, the control of induction is not directly done by cis acting elements on the plasmid, but relies on a compatible host strain.

 

 

ok there is no lac promoter

[Greippi]

ok there is no lac promoter

).

 

But there is in the strain you transform the plasmid into (i.e. the plasmid must be put into e.g. E. coli BL21), So T7 polymerase is induced (the plasmid has a T7 promoter). It says that in the manual I linked you (and as CharonY explained).

Edited February 14, 2011 by Greippi