Composite

Part:BBa_K4658005

Designed by: Tanvi Garg   Group: iGEM23_Stockholm   (2023-10-07)


Anchor Peptide with EGFP

This fusion protein was created as a control for the binding of microplastics - polypropylene using anchor peptide and detection using EGFP. This is a composite part that contains Anchor peptide (BBa_K4658003), AviTag spacer (BBa_K4658001), AviTag Biotinylation Site (BBa_K4658002), TEV Cleavage site (BBa_J18918), Linker Peptide (BBa_K4658000), and EGFP (BBa_K3033009)



Fig.1 Composite part of Anchor peptide (BBa_K4658003) and EGFP (BBa_K3033009)

Usage and Biology

  • The composite part is designed to bind to polypropylene, one of the most common plastic material, and produce a detectable fluorescent signal.(BBa_K4658003)
  • The AviTag can be used for biotinylation by the enzyme BirA and Biotin.(BBa_K4658002)
  • TEV protease is a highly specific enzyme that can cut proteins at the TEV cleavage site without affecting other parts of the protein. Hence, the composite part can also be further cleaved rendering two functional proteins: anchor peptide and EGFP.(BBa_J18918)



Characterization

Agarose Gel

Restriction digestion of the clone was carried out using HindIII and SacI restriction enzymes for both (BBa_K4658004) and (BBa_K4658005). The ligation product was transformed into E. coli Top10 to select and amplify the plasmid for the confirmation via Colony PCR.


Fig.2 Agarose Gel for Colony PCR for clone confirmation of BBa_K4658004 and BBa_K4658005

SDS-PAGE

The expression and purification was carried out in BL21 E.coli, which was induced with 0.5mM IPTG overnight. The 6xHis tag was included to facilitate purification after protein expression and TEV cleavage respectively. The protocols can be found (here). The SDS page for the following is shown below:


Fig.3 SDS-PAGE for purification of Anchor peptide + EGFP (BBa_K4658005)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 224
    Illegal NotI site found at 239
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 199
  • 1000
    COMPATIBLE WITH RFC[1000]


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