Part:BBa_K3739005

Aly01-his-3*Spytag-elastin linker-lecA

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

SpyTag

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.¹

LecA

LecA is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose.²

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 3SpyTag-LecA and further purify it, we added the signal peptide Aly01, followed by a his-tag (6*His) at the N-terminal of 3SpyTag-LecA. We used both BBa_K081005 and BBa_K525998 to construct the expression system and obtained the composite BBa_K3739035 and BBa_K3739115, 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

When we were transferring the plasmid into Vibrio natriegens, colony PCR was used to certify the plasmid was correct. We got the target (1154bp).

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.
2. Kuhaudomlarp, S.; Gillon, E.; Varrot, A.; Imberty, A., LecA (PA-IL): A Galactose-Binding Lectin from Pseudomonas aeruginosa. Methods Mol Biol 2020, 2132, 257-266.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]