Part:BBa_K4613310

pET-29a(+)-T3-YFP

This composite part was constructed to analyze the function of T3-YFP and the association between SpyTag and SpyCatcher. The composite part can be directly imported into plasmid and express T3-YFP at the same time.

SpyTag and SpyCatcher are a pair of reactive protein partners that can spontaneously react to reconstitute the intact folded CnaB2 domain under mild conditions. Hydrophilic elastin-like polypeptides (ELPs) composed of tandem pentapeptides of the form (VPGXG)(n) (where X may be any amino acid except proline) always serve as versatile model systems for biomaterials.

We used ELPs as the backbone of the monomers. Each monomer was fused with 3 SpyTags or 3 SpyCathcers. The polymerization between these two types of monomers can proceed efficiently under multiple conditions. We linked degrading enzymes (M-CPA/ADH3) into the SpyTag monomer to immobilize the enzyme and increase the stability of degrading enzymes.

Fig. 1 Formation of Spy Network. (a) Gene circuit. (b) The polymerization between these two types of monomers.

To verify the combination between T3 and C3, we engineered bacteria expressing T3-YFP (SpyTag-ELPs-SpyTag-ELPs-SpyTag-YFP) and bacteria expressing C3 (SpyCathcer-ELPs-SpyCathcer-ELPs-SpyCathcer). The constructed plasmids were transformed into E. coli BL21 (DE3) and recombinant proteins were expressed using LB medium.

Purified T3-YFP and C3 were subjected to reactions under predefined time and temperature radients. The proteins after reaction were validated by electrophoresis on polyacrylamide gels (SDS-PAGE), followed by Coomassie brilliant blue staining. A distinct target band can be observed at 130 kDa, demonstrating that T3-YFP (62.4 kDa) and C3 (54.5 kDa) are capable of forming the Spy Network (Fig. 2).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 2 Verification of the fabrication between T3-YFP and C3. Lane1: T3-YFP. Lane2: C3. M: Marker. Lane3: T3-YFP and C3(4℃,8 h). Lane4: T3-YFP and C3(4℃,3 h). Lane5: T3-YFP and C3(4℃,1 h). Lane6: T3-YFP and C3(25℃,8 h). Lane7: T3-YFP and C3(25℃,3 h). Lane8: T3-YFP and C3(25℃,1 h). Lane9: T3-YFP and C3(37℃,8 h). Lane10: T3-YFP and C3(37℃,3 h). Lane11: T3-YFP and C3(37℃,1 h).

Reference

1. Dai Z, Yang X, Wu F, et al.Living fabrication of functional semi-interpenetrating polymeric materials[J].Nat Commun,2021, 12 (1): 3422.
2. Zakeri B, Fierer J O, Celik E, et al.Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin[J].Proc Natl Acad Sci U S A,2012, 109 (12): E690-7.
3. Reddington S C, Howarth M.Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher[J].Curr Opin Chem Biol,2015, 29: 94-9.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1007
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1656
  Illegal SapI site found at 75