Luciferase reporter system - choice of promoter?

Sven Even Finborud Borgos svenb at chembio.PLEASEREMOVETHISBEFOREREPLYING.ntnu.no
Tue Nov 12 14:32:19 EST 2002


Hi!

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...) 
Anyone...?


Thanks!

Sincerely,
Sven Even





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