Luciferase reporter system - choice of promoter?
add at yahoo.com
Wed Nov 13 06:31:38 EST 2002
In pSP-luc the T7 promoter is in the opposite sense, so it will transcribe a luc
antisense mRNA, which obviously doesn't encode the luciferase.
What you can try is the expresion from the SP6 promoter which is in the correct
position and sense for the luc gene expression.
Sven Even Finborud Borgos wrote:
> I'm going to do some studies on the expression level of foreign bacterial promoters in E.coli. I'm thinking of using luciferase as reporter system (e.g.
> based on the pSPluc(+) vector from Promega). The reason for choosing luciferase is mainly sensitivity considerations. But I'm wondering what promoter to use
> for my positive control (i.e. a plasmid that will confirm that my luciferase assay is working properly - basically just an inducible promoter upstream of the
> luciferase gene.) I see that Promega has placed a T7 promoter downstream of the reporter gene in the above vector (in a reverse direction), so I'm wondering
> whether to use this one without any further cloning? And simply express the luciferase by using an appropriate strain (with T7 RNA polymerase) and inducing
> with IPTG from the pSPluc(+)-plasmid. Does this sound reasonable - and do any of you have experience with such a scheme?
> Further; is the T7 promoter OK for luciferase expression, or should I use another promoter? (considering thightness, metabolic overload and so on...)
> Sven Even
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