Part:BBa_K2043016:Design
GFP-FBD9
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Fabric Binding Domain 9 was fused downstream in the 5' end of the GFP CDS
FBD 9 was obtained by Phage display technique. We screened for small peptide ligands that selectively bind to the five different fabrics (cotton, wool, silk, linen, polyester) from a library of approximately 10^9 different peptides. These peptides were selected on the target by bio-panning, by which a pool of randomized peptides were screened based on the binding affinity to the target fabric. After three round of bio-panning, a consensus peptide sequence of “FRKKRKS” was found to efficiently bind to polyester. The binding between the “FRKKRKS”-harboring phage and fabric was confirmed by phage ELISA and the dissociation constant was calculated by progressively washing the phage bound target with increasing volume of wash buffer. The Kd was found to be 2*10^-5 M +/- 1*10^-5 M. The binding affinity was further characterized by fusing the FBDs to the N terminal of the GFP.
A linker was introduced in the DNA sequence between FBD9 and the CDS of GFP.
His-tag is added in the C-terminal for protein purification
The sequence was codon optimized for E. coli and restriction sites for the restriction enzymes BpiI, BsaI and BsmBI were avoided. The sequence was confirmed by sequencing and no mutations were observed. The construction was done by Golden Gate technique using BsaI as restriction enzyme to obtain the desired cohesive ends.
Source
GFP-FBD9