Part:BBa_K4613024

pQE-80L-T3-MCPA

The composite part was constructed to find an appropriate expression intensity to achieve balance between metabolic burden and detection efficiency. Moreover, we hope Mature Carboxypeptidase A (M-CPA), which can be found under https://parts.igem.org/Part:BBa_K4613013, can be expressed successfully in E. coli Nissle 1917 (EcN). We tried T5 lac promoter from pQE-80L.

We cloned T3-M-CPA (SpyTag-ELPs-SpyTag-ELPs-SpyTag-Linker-MCPA) into the PQE-80L, constructed pQE-80L-T3 and expressed the recombinant protein in E. coli BL21(DE3) using Terrific Broth medium and 2xYT medium. After incubation at 25℃ overnight or 37℃ for 4h and 8h, respectively, the expression of T3-M-CPA (62.4 kDa) was roughly the same as that of C3. The expression levels of both were very low.

Fig. 1 Results of pQE-80L-T3. (a) The plasmid map of pQE-80L-T3. (b-f) SDS-PAGE analysis of protein expression trials in E. coli BL21(DE3), their expression conditions were TB medium incubated at 37℃ for 4 h, 8 h, 25℃ for 12 h, and 2xYT medium incubated at 37℃ for 8 h, 25°C for 12 h in turn. Lane M: protein marker. Lane 1: induced total protein. Lane 2: precipitation. Lane 3: supernatant.

To degrade Ochratoxin A (OTA) in a more efficient way, we chose two enzymes, Carboxypeptidase A (CPA) and ADH3. We used the methods described by Xiong L et al. (1992) to assay CPA and ADH3 activity. Fig.2 shows that the activity of CPA and ADH3. ADH3 was estimated at approximately 1.939 unit. CPA was estimated at approximately 0.646 unit. These results indicated that ADH3 exhibited 3.0-fold higher activity than CPA.

  Fig. 2 Assay of ADH3 and CPA activity. The reaction mixture containing 290 μl of 25 mM Tris buffer, 500 mM NaCl (pH 7.5), 3.26 mg/mL Hippuryl-L-phenylalanine (HLP), and 10 μl of ADH3 dissolved in 20 mM Tris-HCl (pH 8.0), 10 μl of CPA dissolved in 1 M NaCl (pH 8.4) in eppendorf tube was incubated at 25℃ for 5 min.

Moreover, we used High-Performance Liquid Chromatography (HPLC) to determine the detoxification rate of CPA and ADH3 against OTA. The HPLC chromatograms of degradation products of OTA were shown in Fig. 3. The retention times (RT) of OTA and its degradation product was 1.650 min (CPA), 1.652 min (ADH3) and 0.691 min (CPA), 0.709 min (ADH3). After the treatment of OTA with CPA and ADH3, the peak area of OTA decreased significantly compared with the control group, and the new product appeared at 0.692 min (CPA), 0.709 min (ADH3). The detoxification rates of CPA and ADH3 were 98.9% and 100%. It proved that CPA and ADH3 can degrade OTA to OTα. ADH3 gave a better performance in degrading than CPA because it took less reaction time to degrade OTA completely in higher concentrations.

Fig. 3 High performance liquid chromatography (HPLC) chromatogram retention time of OTA and OTα. a.10 μg/mL OTA after incubation with methanol solution(control). b.HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL M-CPA for 24 h. c. 50 μg/mL OTA after incubation with methanol solution(control). d. HPLC chromatogram of degradation products of OTA after incubation with 5 U/mL ADH3 for 30 min.

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

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

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.
4. Xiong L, Peng M, Zhao M, et al.Truncated Expression of a Carboxypeptidase A from Bovine Improves Its Enzymatic Properties and Detoxification Efficiency of Ochratoxin A[J].Toxins (Basel),2020, 12 (11).

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 1617
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]