Difference between revisions of "Part:BBa K4613310"

Revision as of 08:08, 12 October 2023

pET-29a(+)-T3-YFP

This composite part was constructed to analyze the function of T3-YFP and the association between SpyTag and SpyCatcher. The composite part can be directly imported into plasmid and express T3-YFP at the same time.

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. 1 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.2).This reaction can occur at a variety of temperatures and has good reaction characteristics.

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).

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