Talk:CH391L/S12/Fluorescent Proteins

• Peter Otoupal 19:05, 25 March 2012 (EDT):Are the GFP variants now used in laboratories typically Enhanced GFP, or are the "original" classes still mainly used? Also, how did Zhang et al know to target the Ser65 and Phe64 to improve fluorescence? Or did they just create a library and cross their fingers? Thanks!
  • Midhat Patel 03:56, 26 March 2012 (EDT): The variants used are primarily GFP at this point. The amino acids in locations 64-67 were targeted because random mutation libraries showed that when these were altered, fluorescence either disappeared or was different, so these were the critical amino acids for fluorescence.
    • Peter Otoupal 13:24, 26 March 2012 (EDT):Cool, thanks!

• Joe Hanson 16:50, 19 March 2012 (EDT): Can you add something about Split GFP? It's a pretty cool system for things like solubility evolution, and it allows you to use smaller fusion proteins when compared to attaching the entire GFP ORF.
  • Midhat Patel 03:30, 26 March 2012 (EDT): A section has been added about Split GFP.
  • Joe Hanson 11:55, 26 March 2012 (EDT):Awesome. I know most people use Spkit GFP to engineer more soluble proteins, I think it's a very cool complement system to provide fluorescence. You can effectivly just tag you protein of interest with the GFP11 fragment and have GFP1-10 expressed constitutively. Because the GFP11 tag is so much smaller than full GFP, it's much less likely to interfere with protein function and gives a more physiological phenotype.
    • Midhat Patel 15:09, 26 March 2012 (EDT): Thanks, I added a little more to the section.

• Jeffrey E. Barrick 13:15, 25 March 2012 (EDT): Is there a destabilized GFP that works in bacteria in the iGEM registry? How does it work?
  • Midhat Patel 04:05, 26 March 2012 (EDT): I went through the reporters section in the catalog and none of the descriptions indicated that a destabilized GFP had been used in a part. Searching for articles did not reveal any papers that specifically addressed the production of destablilized GFPs in prokaryotes, although it is obvious that in order for something of that nature to exist, the MODC domain couldn't be used because mouse-specific proteins wouldn't be present in prokaryotes. GFP has been optimized to use bases and amino acids more common in bacteria to enhance expression in prokaryotes.[1]

• Jeffrey E. Barrick 13:20, 25 March 2012 (EDT): This statement under EGFP is confusing "the altered enzymes in EGFP". I believe they are talking about the expression of an enzyme that has been fused to EGFP in this case, since a common application of GFP is to fuse it to a protein to examine localization of that protein in the cell.
  • Midhat Patel 03:36, 26 March 2012 (EDT): I apologize, that's an embarrassing typo. What I meant to say was that the altered amino acids are those that are more common in eukaryotic cells, I have corrected the statement.

• Jeffrey E. Barrick 13:32, 25 March 2012 (EDT): Page could be improved by making the red links go to articles on Wikipedia.
  • Midhat Patel 03:35, 26 March 2012 (EDT): Taken care of, I didn't realize it was linking to non-existent Open WetWare pages and not the Wiki pages themselves.

• Joe Hanson 12:12, 26 March 2012 (EDT):We mentioned this briefly in class, but it's super important to remember that eukaryotic ("firefly") luciferase requires a substrate called luciferin. Luciferase assays in eukaryotes require cell lysis for the most part, since luciferin doesn't diffuse across membranes (although there are some ester modified versions that do better for in vivo assays). In contrast, bacterial luciferase uses flavin, which is present in bacterial cells all the time.

• Adam Meyer 14:09, 26 March 2012 (EDT):How about a mention of mCherry and the other mFruits.
  • Midhat Patel 13:28, 2 April 2012 (EDT): There are a whole lot of various fluorescent proteins that have been developed in different ways, primarily directed mutagenesis of specific amino acids. On the Clontech Fluorescent Protein website you can purchase and read about a lot of different proteins that have been sequences and are commercially available for use as fluorescent markers and reporters.

References

1. Sastalla I, Chim K, Cheung GY, Pomerantsev AP, and Leppla SH. Codon-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria. Appl Environ Microbiol. 2009 Apr;75(7):2099-110. DOI:10.1128/AEM.02066-08 |