Part:BBa_K3739080

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

blrA can be activated by blue light and produce reactive oxygen species(ROS).

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

HutH

The HutH comes from Pseudomonas putida. Under natural conditions, many microorganisms can use the histidine ammonia-lyase (HutH) to change L-histidine into trans-urocanate. It catalyzes the first step in the degradation of histidine and the product, urocanic acid, is further metabolized to glutamate.
HutH are found in the liver of vertebrates and in bacteria such as Escherichia coli, Salmonella and Pseudomonas. It is specific for L-histidine and can be inhibited by D-histidine or imidazole. The active center of the enzyme is thought to be dehydroalanine.

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-HutH and further purify it, we added the signal peptide Aly01, followed by a his-tag (6*His) at the N-terminal of 3SpyTag-HutH. We used both BBa_K081005 and BBa_K525998 to construct the expression system and obtained the composite BBa_K3739068 and BBa_K3739116, which are assembled on the expression vector pET-28a(+) by standard assembly. The progress of this part remains in the stage of design.

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
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 534
  Illegal NgoMIV site found at 970
  Illegal NgoMIV site found at 1705
  Illegal NgoMIV site found at 1941
  Illegal AgeI site found at 607
  Illegal AgeI site found at 1434
  Illegal AgeI site found at 1930
  
• 1000
  COMPATIBLE WITH RFC[1000]