Part:BBa_K3739034

J23100-B0030-Aly01-his-3Spycatcher-B0010

"SpyTag and SpyCatcher are able to form isopeptide bond with the other without any assistant, Aly01 is a signal peptide for secretion, LecA is a sugar binding protein."

Biology

SpyCatcher
SpyTag (a 13-residue peptide) and SpyCatcher (a 116-residue domain) are engineered split fragments of the second immunoglobulin-like collagen adhesin domain (CnaB2) of the fibronectin-binding protein (FbaB) of Streptococcus pyogenes. iGEM team Peking 2016 has modified the peptide and domain into triple SpyTag and triple SpyCatcher, which can form bonds with each other and create Spy Crosslinking Network, enabling the formation of hyper-branched products and polymer network at low concentrations, and gels with a high degree of cross-linkage at high concentrations.

Aly01
Aly01 is alginate lyase from Vibrio natriegens SK42.001, which is secreted out and able to digest alginate to unsaturated alginate oligosaccharide. Its signal peptide (named Aly01 in our parts), which is fused with heterogenous protein, is performed well in E. coli. It is implied that the heterogenous protein fused with Aly01 signal peptide may be also secreted efficiently.

Usage

In order to get secreted 3SpyCatcher and further purify it, we added the signal peptide Aly01, followed by a his-tag (6*His) at the N-terminal of 3SpyCatcher. We used both BBa_K081005 and BBa_K525998 to construct the expression system and obtained the composite BBa_K3739034 and BBa_K3739114, which are assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmids were transformed into Vibrio natriegens, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Characterization

1. Agarose Gel Electrophoresis
After transferring the plasmid into Vibrio natriegens, colony PCR was used to certify the plasmid was correct. The expected result was obtained(2101bp).

Fig.1 The result of colony PCR. Plasmid pET-28a (+).

Reference

1. Yang, X.; Wei, J.; Wang, Y.; Yang, C.; Zhao, S.; Li, C.; Dong, Y.; Bai, K.; Li, Y.; Teng, H.; Wang, D.; Lyu, N.; Li, J.; Chang, X.; Ning, X.; Ouyang, Q.; Zhang, Y.; Qian, L., A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry. ACS Synth Biol 2018, 7 (10), 2331-2339.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1490
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 203