Difference between revisions of "Part:BBa K3002229"
 
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This composite part contains a hygromycin resistance (<a href="https://parts.igem.org/Part:BBa_K3002119">BBa_K3002119</a>) and the WT-PETase with the secretion signal cCA (<a href="https://parts.igem.org/Part:BBa_K3002124">BBa_K3002124</a>) fused with an SP20His-tag for easy purification via Ni-NTA column and enhanced secretion.
 
This composite part contains a hygromycin resistance (<a href="https://parts.igem.org/Part:BBa_K3002119">BBa_K3002119</a>) and the WT-PETase with the secretion signal cCA (<a href="https://parts.igem.org/Part:BBa_K3002124">BBa_K3002124</a>) fused with an SP20His-tag for easy purification via Ni-NTA column and enhanced secretion.
 
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The His-Tag is designed to allow the purification of the WT-PETase and to differentiate between the HA-tagged MHETase and the His-tagged PETase. The PETase will be secreted with the help of the secretion signal ccA. Hygromycin is used as a resistance cassette, to allow a co-transformation.
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<h1> The Chlamy Yummy Project Collection </h1>
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We are proud to present our MoClo part collection for C. reinhardtii - the <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Parts"> Chlamy Yummy project collection</a>.
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These 67 parts are all parts used during our project and were specifically designed and codon optimized for Chlamydomonas.  Among them are basic parts (L0) of a novel mutant of the PETase (<a href="https://parts.igem.org/Part:BBa_K3002014">BBa_K3002014</a>), the wildtype PETase and MHETase as well as a variety of functional composite parts (L1+2). Containing different tags as well as selection markers, this collection serves as a perfect base for plastic degradation projects with Chlamydomonas. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. 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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Visit our <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Parts">parts site</a> to get an overview over all parts.
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<!-- Add more about the biology of this part here
 
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Latest revision as of 00:01, 14 December 2019


L2 hygromycin resistance + cCA_WT-PETase_SP20His

This composite part contains a hygromycin resistance (BBa_K3002119) and the WT-PETase with the secretion signal cCA (BBa_K3002124) fused with an SP20His-tag for easy purification via Ni-NTA column and enhanced secretion.

The His-Tag is designed to allow the purification of the WT-PETase and to differentiate between the HA-tagged MHETase and the His-tagged PETase. The PETase will be secreted with the help of the secretion signal ccA. Hygromycin is used as a resistance cassette, to allow a co-transformation.

The Chlamy Yummy Project Collection

We are proud to present our MoClo part collection for C. reinhardtii - the Chlamy Yummy project collection.

These 67 parts are all parts used during our project and were specifically designed and codon optimized for Chlamydomonas. Among them are basic parts (L0) of a novel mutant of the PETase (BBa_K3002014), the wildtype PETase and MHETase as well as a variety of functional composite parts (L1+2). Containing different tags as well as selection markers, this collection serves as a perfect base for plastic degradation projects with Chlamydomonas. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. 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:

Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.

Level 1 parts are combinations of basic parts and usually form functional transcription units.

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.

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. After trying MoClo once, you won’t go back to traditional ligation. It is incredibly easy and reliable. Visit our parts site to get an overview over all parts.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 2868
    Illegal PstI site found at 3841
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2048
    Illegal PstI site found at 2868
    Illegal PstI site found at 3841
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1408
    Illegal XhoI site found at 250
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2868
    Illegal PstI site found at 3841
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2868
    Illegal PstI site found at 3841
    Illegal NgoMIV site found at 773
    Illegal NgoMIV site found at 955
    Illegal NgoMIV site found at 1326
    Illegal NgoMIV site found at 2603
    Illegal NgoMIV site found at 2630
    Illegal NgoMIV site found at 4332
  • 1000
    COMPATIBLE WITH RFC[1000]