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Gram positive RBS + GFP not expressed in pgem-T within Dh5alpha

gram positvie RBS GFP pgem-t Dh5alpha

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#1 thefallengrace

thefallengrace

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Posted 10 May 2014 - 09:01 AM

Hi there,

 

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? 

 

 







Also tagged with one or more of these keywords: gram positvie, RBS, GFP, pgem-t, Dh5alpha

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