Part:BBa_K3739068

J23100-B0030-Aly01-his-3Spytag-elastin linker-hutH-B0010

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

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.

hutH

HutH exists in various microorganisms in nature that could catalyze L-histidine to trans-urocanate (Fig. 1). In our project, the gene of HutH from Pseudomonas putida was expressed in chassis bacteria to produce histidine ammonia-lyase (HutH). After that, the product of trans-urocanate was further converted to glutamic acid.
HutH is found in the livers of vertebrates and bacteria, such as E. coli, Salmonella and pseudomonas, which is specific to L-histidine and can be inhibited by D-histidine or imidazole.

Fig. 1. Schematic diagram of HutH catalytic process.

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 598
  Illegal NgoMIV site found at 1034
  Illegal NgoMIV site found at 1769
  Illegal NgoMIV site found at 2005
  Illegal AgeI site found at 671
  Illegal AgeI site found at 1498
  Illegal AgeI site found at 1994
  
• 1000
  COMPATIBLE WITH RFC[1000]