Part:BBa_K4613012

C3

This part codes for hydrophilic elastin-like polypeptides (ELPs) fused with triple SpyCatcher sequences, through polymerization by covalent bonding between SpyTag and SpyCatcher, forming polymeric scaffolds. If you want to learn about the detailed introduction of ELPs, you can click the link below. https://parts.igem.org/Part:BBa_K4613010

We first cloned C3 into the PQE-80L , constructed PQE-80L-C3 and expressed the recombinant protein in E. coli BL21(DE3) using Terrific Broth medium and 2xYT medium. After incubation at 20℃ overnight or 37℃ for 4h, respectively, we found that C3 expression level in the supernatant was very low, and no obvious bands were found at 54.5KDa As shown in Fig. 2b-c. Considering the weak strength of the T5 promoter, we cloned C3 into a vector containing a stronger T7 promoter.

To verify the sIPN system, 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.4).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 1 Diagram of OTA degradation principle.

Fig. 2 Results of pQE-80L-C3. a. The plasmid map of pQE-80L-C3. b.SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3) cultured in Terrific Broth medium overnight using pQE-80L-C3. The temperature was 20℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant. c. SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3) cultured in Terrific Broth medium for 4 hours using pQE-80L-C3. The temperature was 37℃. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitate. Lane 3: supernatant.

  Fig. 3 Results of pET-29a(+)-C3. a. The plasmid map of pET-29a(+)-C3. b. SDS-PAGE analysis of the purified protein C3 in E. coli BL21(DE3) cultured in LB medium express protein for 3 hours at 37℃. Lane M: protein marker. Lanes 1-7: flow through and elution containing 20, 50, 50, 100, 100, 250, 250 mM imidazole, respectively.

Fig. 4 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).

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 XhoI site found at 1600
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]