what do you know about GFP?

dalex at NEXUS.MICROIMM.MCGILL.CA dalex at NEXUS.MICROIMM.MCGILL.CA
Tue Jun 17 15:01:10 EST 1997


Dear BioNetters

I am a graduate student preparing to construct a GFP fusion
to an E.coli protein. The scientific literature and biotech
catalogues are overflowing with GFP vectors, colour shifted
GFP mutants and GFP fusion systems that claim to be
effective. What I would like is some practical advice
on constructing GFP fusions in E. coli.

If you have constructed such a fusion - and were satisfied
with the results, I would really appreciate it if you 
would take the time to respond to my questions.

1) What system did you use?
   (i.e. Was it a kit?  From a journal reference? Something
    you developed/modified yourself?)
2) What did you like or not like about it? 
3) What kind of GFP did you use and why?
4) Did you use a special strain of E. coli?
5) Did you construct an N- or C-terminal fusion?
   (or if you did both, was one better than the other?)
6) How did you assay GFP activity?
   (i.e. FACS? confocal microscopy? hand held UV?)
7) Is there anything else I should know?

Many thanks to anyone who can help.


David Alexander.






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