i need some help here, im quite new in protein expression so here goes my question.
i have cloned GFP + RBS into pgem-T. The key here is, the RBS come from a gram positive bacteria, with 13 bp spacer.
I was hoping to see my dh5alpha fluorescence , but none of my transformants shows any kind of fluorescence.
So i repeat again, by cloning GFP WITHOUT RBS into pgem-t, and some of the transformants fluorescent
Didn't pgem-T have T7 and Sp6 promoter?
or does my G + RBS is spoiling the protein translation, hence giving me a frameshift resulting no gfp protein was produce
perhaps e.coli doesnt recognize the rbs?