Part:BBa_K3075002:Design

TycA^S563A-SpyT-His

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2289
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2299
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1223
  Illegal BsaI.rc site found at 2143
  Illegal SapI.rc site found at 2499
  

Design

The following gene construct was designed to enable the recombinant expression of the TycAS653A protein within an E. coli chassis (Figure 3). Gibson forward and reverse overhangs were added the 5’ and 3’ ends for cloning via Gibson Assembly. The SpyTag sequence was added to the C-terminal to enable the conjugation of the TycAS653A protein to SpyCatcher containing alpha-prefoldin arms of the assemblase scaffold. A hexa-histidine tag was added to the C-terminal end to enable the purification of the protein once expressed by immobilised metal affinity chromatography (IMAC) via a nickel-NTA column. Each gene component was separated by a GSG linker sequences to increase the flexibility between each peptide and prevent steric hindrance of protein folding.

Image

Figure 1: Sequence annotation of TycAS653A-SpyT-His gBlock contains the TycA gene (orange) with mutation S653A (yellow), SpyTag (pink) and a hexahistidine tag (grey) separated by GSG linkers (silver), TAA stop codon (brown) enclosed within gibson forward and reverse overhangs (orange).

Source

Originated from Brevibacillus parabrevis