Coding

Part:BBa_K1321371

Designed by: Xenia Spencer-Milnes   Group: iGEM14_Imperial   (2014-10-17)

Linker-CBDcipA-Linker + sfGFP with LacI promoter

A LacI-promoter expression construct of super-folder GFP fused C-terminally to CBDcipA (a cellulose-binding domain), which contains an endogenous N and C-terminal linker sequence.

At present the site-directed mutagenesis for this construct is in progress to correct an illegal EcoRI site which was identified in the CBDcipA.

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 stop codon plus 6 bp at the end of the sequence are included the RFC25 suffix which is not shown. The prefix to this part is RFC10 format.

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 (protocol here) – it was determined that the CBDcipA-sfGFP fusion had the greatest binding ability in comparison to the four other CBDs fused to sfGFP after three washes with 5% BSA.

In the same assay, results suggested that CBDcipA that on average overall it had the second best ability to bind bacterial cellulose, as shown by the washes with PBS, 70% EtOH and dH2O.

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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 383
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 383
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 383
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 383
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 383
    Illegal NgoMIV site found at 230
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 962


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Parameters
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