Coding

Part:BBa_K1321337

Designed by: Chris N Micklem   Group: iGEM14_Imperial   (2014-10-08)

sfGFP in Freiburg format (RFC 25)

sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins. We used the part I746909 as our template for PCR with primers that use overhanging ends to insert the Freiburg sites.

Usage and Biology

Superfolder Green Fluorescent Protein (sfGFP) is a mutant of mut3b GFP (E0040) with improved in vivo properties. It was first entered into the registry by the Cambridge iGEM team in 2008; please see I746916 part description and http://openwetware.org/wiki/IGEM:Cambridge/2008/Improved_GFP for detailed information about the properties of sfGFP.


IC-2014 glow1.jpg

IC-2014 glow2.jpg


We've successfully used this Freiburg sfGFP as a reporter. As part of our project, we carried out an assay to determine the relative binding ability of CBD-sfGFP fusions to bacterial cellulose. The Freiburg sites made it easier to create fusions between our CBDs with this reporter sfGFP, and its superfolding nature gave intense fluorescence even when fused with and without linkers.


In our first assay performed to determine the relative strengths of various CBDs’ binding to bacterial cellulose – represented by the percentage fluorescence left from CBDs fused to (sfGFP (RFC25)) bound to bacterial cellulose discs, when subjected to various washes - it was determined that the fusion with dCBD had the greatest binding ability in comparison to four other CBDs fused to sfGFP after three washes with both dH2O and 70% EtOH.

In the same assay, results suggested that dCBD had on average the third greatest ability to bind bacterial cellulose, after three washes with both PBS and 5% BSA.

IC14 - dH2Obplot1.png
IC14-EtOHbplot1.png
IC14-PBSbplot1.png
IC14-BSAbplot1.png


In summary, this sfGFP served its purpose.



Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/reporter/gfp
//function/reporter/fluorescence
Parameters
colorGreen