3 replies to this topic

[mnqcljsm]

Dear all,

This may be more of a molecular biology question...

I want to use a reporter plasmid to look for activation of a particular gene promoter in E. coli. I plan to clone the promoter sequence for the GOI into a plasmid upstream of a reporter gene (either GFP or beta-gal i havent decided yet).

Please could anyone recommend suitable plasmids for doing this. pUC18 for example could be suitable for a beta-gal reporter but Im not sure of a suitable GFP containing vector that i could use.

What will be the impact of plasmid copy number? - i assume that high copy number would give me a stronger signal upon activation of the promoter?

All the best,

mnq

[macroman]

Please see the following attachment.

Hope this helps

Attached File(s)

•  paper_for_bacterial_expression.PDF (365.14K)
  Number of downloads: 23

[mnqcljsm]

many thanks!

[pDNA]

I think using the pGFPmut3.1 promoter should be suitable ...delete the lac promoter and replace it with your promoter of choice!

If you are going to screen for promoter-activity on agar-plates the higher the copy number the better because you will be able to find high-level expression clones by eye (even without UVlight).

Regards,
p