Coding

Part:BBa_K1321348

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

sfGFP fused to dCBD with linker in RFC 25

Super-folder GFP fused N-terminally to dCBD (a cellulose-binding domain), which contains an endogenous N-terminal linker sequence.

This construct is part of a library of Super-folder GFP fusions with cellulose binding domains, which we used to assay the CBD binding affinity. Please see our project page for more information. The collection of sfGFP-CBD fusion parts can be seen in the table below: IC14-sfGFP-part-table.PNG

Note that the start and stop codon, plus 6 bp either side of the sequence, are included the RFC25 prefix and suffix which is not shown.

As part of our project we carried out an assay to determine the relative binding ability of CBD-sfGFP fusions to bacterial cellulose.

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


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
//binding/cellulose
//cds/reporter/gfp
Parameters
None