Difference between revisions of "Part:BBa K3002004"
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<h1> The Kaiser Collection </h1>
 
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We are proud to present our very own MoClo part collection for C. reinhardtii - the <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Part_Collection">Kaiser collection</a>.
 
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These 20 Parts are specifically designed and codon optimized for Chlamydomonas. Among them are regulatory elements, antibiotic resistances, resistance cassettes, secretion signals and tags. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. With these, expression and secretion in Chlamy will be a success. Because this is a MoClo collection, the parts are highly standardized for worldwide application. The combination with other part collections works fast and easy. While in MoClo, nomenclature is a bit different from the iGEM BioBricks, it is quickly explained:
 
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Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
 
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Level 1 parts are combinations of basic parts and usually form functional transcription units.
 
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Level 2 parts are combinations of Level 1 parts, in case you want to transfer multiple transcription units at once. For example, you can pair your gene of interest with a selection marker.
 
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The great thing about the Kaiser Collection and MoClo is that the ligation works in a one pot, one step reaction, as the Type IIs restriction enzymes cut out their own recognition sites. This way, multiple constructs can be combined linearly in a fixed order to create complex structures. This is ensured by the standardized overlaps that assign the parts one of 10 positions in the final constructs.
 
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After trying MoClo once, you won’t go back to traditional ligation. It is incredibly easy and reliable.
 
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For this reason, we believe that our Kaiser Collection will strike a significant chord, as the future lies in standardized, easy to use methods such as MoClo.
 
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Visit our <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Part_Collection">part collection site</a> to get an overview over all parts of the Kaiser Collection
 
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Revision as of 15:54, 10 December 2019


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]