Part:BBa_K2043013

GFP-FBD6

In the context of Paris Bettencourt 2016 iGEM project, Frank&Stain, we looked for fabric binding domains with phage display in order to optimise our enzymes on the topic of pigment degradation. More information can be consulted in the wiki, but we wanted to degrade wine stains, and we developed the FBD in order to direct the enzymatic activity to the fabric.

We fused the GFP gene with the FBD6 (in the N-terminal of the GFP) in order to understand two things: (i)if the GFP fluorescence would be affect by the binding of the FBD and (ii)if the binding capacity would be affected.

The results for FBD6-GFP are as follows:

FBD-6 has a sequence of MRLSVPN, has medium to low specificity to all fabrics in most conditions. It exhibits high specificity to wool in catechol and linen in PBS. This amino acid sequence has a positive charge, and a Hydropathicity (GRAVY): index of -0.071 (not very hydrophilic).

These experiments were performed by obtaining cell extract of cells of a strain of E. coli<i> overexpressing FBD6-GFP, incubating the cell extract with the fabrics overnight, and then performing 2 washes with the indicated solutions.

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]

GFP-FBD6

Jain, P., Soshee, A., Narayanan, S. S., Sharma, J., Girard, C., Dujardin, E., & Nizak, C. (2014). Selection of arginine-rich anti-gold antibodies engineered for plasmonic colloid self-assembly. The Journal of Physical Chemistry C, 118(26), 14502-14510.

Soshee, A., Zürcher, S., Spencer, N. D., Halperin, A., & Nizak, C. (2013). General in vitro method to analyze the interactions of synthetic polymers with human antibody repertoires. Biomacromolecules, 15(1), 113-121.

Boyer, S., Biswas, D., Soshee, A. K., Scaramozzino, N., Nizak, C., & Rivoire, O. (2016). Hierarchy and extremes in selections from pools of randomized proteins. Proceedings of the National Academy of Sciences, 201517813.