Hello everyone, I am Achmad. I hope you all in good conditions.
Currently, we will work in Recombinant protein with expression host in bacteria (E.coli BL21 DE3).
We have done construct our pET28a bacterial expression plasmid, and our gene of interest is cloned into the NdeI-XhoI restriction site. My supervisor suggests adding the GFP reporter gene to my construct. He said that the GFP reporter is essential for expression control. In my view, this fusion with GFP reporter could disrupt the function of protein interest. Could you give your opinion about this? Please give me the references (articles and/or related) if GFP is necessary or not for bacterial host protein expression. And where is the best position for the GFP reporter's sequence if it is included in my plasmid? (If the GFP is necessary).
Here I also attach our plasmid map, Sorry some of our labels are censored.
Thank you very much for your help.
a lot of time in the lab is spent cloning, so if you can clone as many as different versions of your cloning product the better. You can clone your gene without GFP, with GFP in the N-terminus, in the C-terminus, you can add epitope tags, etc. It's best to do it in a single cloning effort, at the end you'll have very valuable reagents without wasting time.
Depending on your protein, you can clone the GFP in multiple areas of your gene as Oscar L. Sierra suggested. Have a look at these previous threads - they provide a lot of information:
https://www.researchgate.net/post/Is-there-any-interference-of-GFP-tag-on-protein-structure-and-function
https://www.researchgate.net/post/Interference-of-GFP-tag-on-protein-structure-function
Good luck!
Oscar L. Sierra's opinion is good. If you are worried whether GFP might interfere with the protein's function, you can add a flexible Gly/Ser based linker. Depending on the target protein, varying the length and type of linkers usually helps to keep the reporter from interfering with the protein's structure/function.
Ref:
Article Tuning the Flexibility of Glycine-Serine Linkers To Allow Ra...
Plus can you elaborate on how adding GFP tag helps in control of the protein's expression in bacteria ?
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