Part:BBa_K3002014

Mutant PETase for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of a mutant version of the PETase (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii. The following mutations were inserted to increase activity: R280A, S238F and W159H (Joo, S. et al., 2018) (Austin, H. et al, 2018). Combined with a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1-3 of RBCS2, it perfectly matches the part BBa_K3002027 (pAR promoter A1-B2), resulting in a high expression (Eichler-Stahlberg et al., 2009). To detect or purify the target protein a tag of the Kaiser Collection like BBa_K3002010 (sp20 HA-tag), BBa_K3002017 (HA-tag), BBa_K3002018 (sp20 His-tag), BBa_K3002028 (His-tag) is recommended. A secretion signal (BBa_K3002007 (cCA), BBa_K3002008 (GLE) or BBa_K3002009 (ARS)) can be added, when using the part BBa_K3002003 (pAR promoter (A1-A3)) as a promoter.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 507
  Illegal PstI site found at 1480
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 507
  Illegal PstI site found at 1480
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 507
  Illegal PstI site found at 1480
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 507
  Illegal PstI site found at 1480
  Illegal NgoMIV site found at 242
  Illegal NgoMIV site found at 269
  
• 1000
  COMPATIBLE WITH RFC[1000]