Difference between revisions of "Part:BBa K3002101"
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This composite part contains the PAR-promoter (<a href="https://parts.igem.org/Part:BBa_K3002027">BBa_K3002027</a>) in combination with the RPL23-Terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>), an HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) and the coding sequence of the mutant PETase, Mutate M, of the iGEM team TJUSLS China (<a href="https://parts.igem.org/Part:BBa_K1921003">BBa_K1921003</a>) plus the MoClo connectors for positions B2-B3 ( <a href="https://parts.igem.org/Part:BBa_K3002303">BBa_K3002303</a>), B4-B5 ( <a href="https://parts.igem.org/Part:BBa_K3002304">BBa_K3002304</a>) and B5-B6 (<a href="https://parts.igem.org/Part:BBa_K3002305">BBa_K3002305</a>).  
 
This composite part contains the PAR-promoter (<a href="https://parts.igem.org/Part:BBa_K3002027">BBa_K3002027</a>) in combination with the RPL23-Terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>), an HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) and the coding sequence of the mutant PETase, Mutate M, of the iGEM team TJUSLS China (<a href="https://parts.igem.org/Part:BBa_K1921003">BBa_K1921003</a>) plus the MoClo connectors for positions B2-B3 ( <a href="https://parts.igem.org/Part:BBa_K3002303">BBa_K3002303</a>), B4-B5 ( <a href="https://parts.igem.org/Part:BBa_K3002304">BBa_K3002304</a>) and B5-B6 (<a href="https://parts.igem.org/Part:BBa_K3002305">BBa_K3002305</a>).  
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<img src="https://2019.igem.org/wiki/images/c/cb/T--TU_Kaiserslautern--Goldkriteriumhoffentlich.png"/>
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<p class="caption"><span class="phat">Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas</span>
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                (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control. 
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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/Part_Overview"> 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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</p>
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<p>
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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/Part_Overview">parts site</a> to get an overview over all parts.
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Revision as of 19:12, 13 December 2019


Level 1 Mutant PETase + HA (Phytobrick)

This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), an HA-tag (BBa_K3002017) and the coding sequence of the mutant PETase, Mutate M, of the iGEM team TJUSLS China (BBa_K1921003) plus the MoClo connectors for positions B2-B3 ( BBa_K3002303), B4-B5 ( BBa_K3002304) and B5-B6 (BBa_K3002305).

Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.

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
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1722
  • 1000
    COMPATIBLE WITH RFC[1000]