Part:BBa_K1321362

sfGFP fused to CBDcex driven by LacI

A LacI-promoter expression construct of super-folder GFP fused N-terminally to CBDcex (a cellulose-binding domain).

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 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.

The binding ability of this CBD to bacterial cellulose was characterised when fused to sfGFP, relative to other CBDs fused to sfGFP. The binding ability was represented by the percentage fluorescence remaining from the sfGFP-CBD fusions bound to bacterial cellulose discs, when subjected to various washes (protocol [http://2014.igem.org/Team:Imperial/Protocols here]). When washed with PBS, sfGFP-CBDcex fusions performed the best out of all the CBDs tested (third graph) followed by washes with 5% BSA (fourth graph), dH20 (first graph) and 70% EtOH (second graph).

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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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 230
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 245