Part:BBa_K4613015

T3-YFP

T3 (BBa_K4613011) and C3 (BBa_K4613012) can form protein complexes by elastin-like polypeptides (ELPs) monomers containing SpyTags and SpyCatchers.

Different functional proteins can be incorporated into the polymeric scaffolds in a flexible manner due to its programmability. In this part, NAU-CHINA 2023 incorporated Yellow Fluorescent Protein (YFP). We fused YFP into T3 to immobilize the fluorescent protein and test the combination between T3 and C3.

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.

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℃, 8h). Lane4: T3-YFP and C3(4℃, 3h). Lane5: T3-YFP and C3(4℃, 1h). Lane6: T3-YFP and C3(25℃, 8h). Lane7: T3-YFP and C3(25℃, 3h). Lane8: T3-YFP and C3(25℃, 1h). Lane9: T3-YFP and C3(37℃，8h). Lane10: T3-YFP and C3(37℃, 3h). Lane11: T3-YFP and C3(37℃, 1h).

We mixed the bacteria producing T3-YFP and the bacteria producing C3, and prepare pellets with 1.5% sodium alginate solution to wrap them. And then the immobilized microcapsules were incubated in 1.5 mL of chitosan solution (0.4% (w/v)) for 15 minutes, while gently shaking and adding a layer of chitosan shell to the microcapsules(Fig. 3 a-1.). At the same time, we also attempted to directly use purified protein T3-YFP to produce the above-mentioned microcapsules(Fig. 3 a-2.). After placing two types of immobilized microcapsules at room temperature for 12 hours, we found that the microcapsules containing T3-YFP exhibited significant yellow fluorescence outside the microcapsules, proving that proteins can leak out of the immobilized microcapsules(Fig. 3 b-1.). On the contrary, the microcapsules containing engineered bacteria exhibit brighter yellow fluorescence only in the microcapsules, without any leakage(Fig. 3 b-2.).This means that the engineered bacteria and sIPN system will not leak, but small molecules of proteins will leach out of the microcapsules.

Fig. 3 Image of immobilized microcapsules. a-1. Immobilized microcapsules containing engineered bacteria with T3-YFP and C3 (0h); a-2. Immobilized microcapsules containing purified T3-YFP protein (0h); b-1. Immobilized microcapsules containing engineered bacteria with T3-YFP and C3 (12h); b-2. Immobilized microcapsules containing purified T3-YFP protein (12h); Scale bar: 1 cm.

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 940
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1589
  Illegal SapI site found at 8