Part:BBa_K3075003:Design

LXYL-P1-2- SpyT-His

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1062
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1462
  Illegal PstI site found at 1499
  Illegal NgoMIV site found at 168
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design

The following gene construct was designed to enable the cloning and expression of the recombinant protein LXYL-p1-2-SpyT within a T7 expression system.

Additions to the gene are as follows:

• Gibson forward and reverse overhangs – complementary overhangs outside the protein coding region enables cloning into pET19b via Gibson assembly
• Hexahistidine peptide tag – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.

Additionally, GSG linkers are included between the peptide sequences. This flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag).

Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the LXYL-p1-2-SpyT gene construct. Image by Linda Chen.

Source

Originated from Lentinula edodes (Shiitake mushroom)