Difference between revisions of "Part:BBa K1321346"

Latest revision as of 05:50, 2 November 2014

CBDclos fused to sfGFP in RFC 25

super-folder GFP fused C-terminally to CBDclos (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 [http://2014.igem.org/Team:Imperial/Functionalisation project page] for more information. The collection of sfGFP-CBD fusion parts can be seen in the table below:

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 and sfGFP fusions to bacterial cellulose.

Our first assay was performed to determine the relative strengths of various CBDs’ binding to bacterial cellulose. These were represented by the percentage fluorescence left from CBDs fused to (sfGFP (RFC25)) bound to bacterial cellulose discs, when subjected to various washes. Full protocol can be found ([http://2014.igem.org/Team:Imperial/Protocols here])

In the same assay, results suggested that dCBD, CBDcex, CBDcipA, and C-terminal CBDclos (all fused to sfGFP) had greater ability to bind bacterial cellulose than N-terminal CBDclos. Three washes with dH2O, PBS and 5% BSA were carried out to monitor binding strength. It was determined that the fusion with N-terminal CBDclos was, on average, our fifth strongest CBD, in comparison other CBDs tested.

Figure 1 - CBD binding strength after three dH2O washes.

Figure 2 - CBD binding strength after three 70%Ethanol washes

Figure 3 - CBD binding strength after three PBS washes

Figure 4 - CBD binding strength after three BSA washes