Part:BBa_K4613025

pQE-80L-C3

The composite part was constructed to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency. Moreover, we hope our C3, which can be found under https://parts.igem.org/Part:BBa_K4613012, can be expressed successfully in E. coli Nissle 1917 (EcN). We tried T5 lac promoter from pQE-80L.

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. 3 b-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.

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 BglII site found at 1081
  Illegal BamHI site found at 1026
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1191
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 94