Part:BBa_K4613301

pET-29a(+)-C3

This composite part was constructed to analyze the function of C3 and the intensity of the T7 lac promoter. The composite part can be directly imported into plasmid and express induced C3 with IPTG.

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

 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 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 54.5 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.

<p style="text-align: center!important;"><b> 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: precipitation. 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: precipitation. 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
  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]