Coding

Part:BBa_K3002004

Designed by: Dorothée Klein   Group: iGEM19_TU_Kaiserslautern   (2019-10-12)
Revision as of 15:51, 10 December 2019 by Tscholak (Talk | contribs)


Wildtype PETase for Chlamydomonas reinhardtii (Phytobrick)


The WT-PETase was expressed in C.reinhardtii and little secretion was shown with the secretion signal cCA in combination with the SP20 tag. Secretion in combination with the SP20 tag was detected. Compared to the Mut-PETase it has no point mutations. The WT-PETase is important to judge the effect of the point mutations in the Mut-PETase regarding their effect onto the efficiency of the enzyme.

Overview of different level 2 MoClo constructs. We designed 35 different level 2 constructs by using the modular cloning system (MoClo) and transformed these into Chlamydomonas reinhardtii. These constructs contain promoters (PPSAD, PAR, PTub2), terminators (PSADter, RPL23ter, Tub2ter), and the coding sequences for selection markers (aadA, Hygro), tags (HA, His, SP20-HA, SP20-His), secretion signals (cCA, ARS, GLE) and the enzymes MHETase, wild-type PETase (WT-PETase), mutated PETase (Mut-PETase) and the mutated PETase from the iGEM team TJUSLS China 2016 (Mutate M).

This basic part contains the coding sequence of the wildtype PETase (B3-B4). 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]


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Parameters
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