GFP fused proteins - i'm going to cry... (Aug/20/2010 )
I'm trying to use GFP as a reporter gene to show arsenic in E.coli.
I cloned GFP after another gene which has a sensitive promoter to arsenic and expected to see fluorscent bacteria after adding arsenic compounds to media. but the results were not be good.the expression of GFP is not enough.i repeated the the experiment again and again...and ...nothing!!!
i'm really upset. i think the genes sequences are ok and the problem is about the expression.help me pls
If you fused the gene to GFP, the protein may interfere with GFP folding. An alternative would be to fuse only the gene promoter to GFP rather than the whole gene. The promoter will be active in the presence of arsenic, resulting in expression of GFP.
Did you fuse the GFP before the stop codon of the first gene?
Was the fusion integrated into the genome or was it expressed from a plasmid?
You can get better signal if you use Venus instead of eGFP. That should be 5-10 times brighter.
you could clone just GFP downstream of your arsenic inducible promotor, rather than making a fusion protein. Remember to include a ribosomal binding site, this could improve the signal if its the fusion protein's folding thats problematic and should still indicate induction of the promotor