Construction of a N-terminal GFP fusion plasmid
Posted 17 March 2011 - 09:46 PM
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?
Posted 25 March 2011 - 06:00 PM
Posted 25 March 2011 - 11:02 PM
maybe something is wrong with your expression unit? ...can you rule out that? ...ribosome binding site positioned correctly? spacing between RBS and ATG correct?