Part:BBa_K4613301

pET-29a(+)-C3

The part use pET-29a(+) vector to express C3 to test its function and find its proper intensity. C3, fused hydrophilic elastin-like polypeptides (ELPs) 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 used ELPs as the backbone of the monomers. Each monomer was fused with 3 SpyTags or 3 SpyCatchers. The polymerization between T3 and C3 can proceed efficiently under multiple conditions.

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

We predicted the structure of C3. We have to rely on I-TASSER's online server to perform structure prediction on C3 using the folding recognition method. We select the result that is ranked first as our prediction.

Fig. 2 C3 protein predected by I-TASSER.

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 4 h, respectively, we found that C3 expression level in the supernatant was very low, and no obvious bands were found at 54.5 kDa As shown in Fig. 3b-c. Considering the weak strength of the T5 promoter, we cloned C3 into a vector containing a stronger T7 promoter.

Fig. 3 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.

We cloned C3 into the pET-29a(+)(Fig. 2a), and expressed it in E. coli BL21(DE3) using LB medium.

After overnight incubation at 20℃, C3 (54.5 kDa) was determined to be soluble and purified on a HiTrap Ni-NTA column. The purified protein was verified by SDS-PAGE. After that, obvious target bands can be seen at 54.5 kDa, confirming the successful expression of C3 in pET-29a(+) vector as shown in Fig 4b (lanes 8 and 9).

  Fig. 4 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.

To verify the combination between T3 and C3, we engineered bacteria expressing T3-YFP (SpyTag-ELPs-SpyTag-ELPs-SpyTag-YFP) and bacteria expressing C3 (SpyCatcher-ELPs-SpyCatcher-ELPs-SpyCatcher). 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. 5).This reaction can occur at a variety of temperatures and has good reaction characteristics.

Fig. 5 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).

Using T3 and C3, the formation of Semi-interpenetrating polymer network (sIPN) leads to strengthening of the mechanical property of the proteins and the versatile functionalization of the scaffold polymer by incorporating ADH3. We hope that this part and BBa_K4613303 can be associated together to make sIPN immobilized microcapsules, which can degrade OTA in wine production factory in a efficient, sustainable, and environmentally-friendly way.

Fig. 1 immobilized microcapsules for Encapsulation of Engineered E. coli.

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
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1645
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1620
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]