Coding

Part:BBa_K2043008:Design

Designed by: Mislav Acman   Group: iGEM16_Paris_Bettencourt   (2016-10-13)


bpuI-FBD1 laccase codon optimized for E.coli


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 699
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Fabric Binding Domain 1 was fused downstream in the 5' end of the original bpul CDS
FBD 1 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 “MPRLPPA” was found to efficiently bind to different fabrics. The binding between the “MPRLPPA”-harboring phage and fabrics's was confirmed by phage ELISA and the binding affinity was further characterized by fusing the FBDs to the N terminal of the GFP. linker was introduced in the DNA sequence between FBD1 and the CDS of bpul.
His-tag is added in the C-terminal for protein purification
This BioBrick is the bpul gene (Bba_K2043007) and 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

Bacilus pumillus

References

"Identification and characterization of a cellulose binding heptapeptide revealed by phage display." Guo, Jing, et al., Biomacromolecules 14.6 (2013): 1795-1805.

"Screening for cellulose nanowhiskers binding peptides by phage display." Guo, Jing, et al. 2010 Pittsburgh, Pennsylvania, June 20-June 23, 2010. American Society of Agricultural and Biological Engineers, 2010.