Construction of a N-terminal GFP fusion plasmid - No detectable GFP fluorescence (Mar/18/2011 )
I have been trying to make a GFP fusion construct (N-terminal). So basically i fused the 5'UTR region of the gene of interest and fused in to GFP in frame under a pBAD promoter. Both the pBAD promoter and the GFP protein was already available in the starting plasmid. All i did was PCR amplify the region of interest and inserted between the promoter and GFP. The plasmid was sequenced and it seems fine. But when i induced for GFP expression (arabinose) i don't see GFP expression compared to un-induced cells. I really don't what to make of it. Has anyone experienced this type of problem before? What could be the problem?
Did you check where the protein localises? I mean if you have a clue from its signal peptide. I maybe wrong, but you may not see anything if it's transported into cytosol and that is the big problem of GFP.
it is possible that your fusion partner prevents the GFP from correct folding ...you did SDS-PAGE or western to confirm your fusion protein is made upon induction?
maybe something is wrong with your expression unit? ...can you rule out that? ...ribosome binding site positioned correctly? spacing between RBS and ATG correct?