Difference between revisions of "Part:BBa K3002208"
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This composite part contains a spectinomycin resistance (<a href="https://parts.igem.org/Part:BBa_K3002102">BBa_K3002102</a>) and the Mutant PETase (<a href="https://parts.igem.org/Part:BBa_K3002108">BBa_K3002108</a>) fused with an HA-tag for easy detection via HA-antibody and with the secretion signal ARS.
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The construct encodes the secretion signal ARS in front of MUT-PETase gene. As selection marker an aadA cassette is used. Constructs encoding the ARS show a higher secretion than constructs encoding an cCa or GLE secretion signal upstream to the MUT-PETase. The MUT-PETase is crucial for the degradation of PET into terephthalic acid and ethylene glycol.
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===Usage and Biology===
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<div class="figure">
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<img src="https://2019.igem.org/wiki/images/b/b8/T--TU_Kaiserslautern--resultsFigure1.svg"/>
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<p class="caption"><span class="phat">Overview of different level 2 MoClo constructs.             
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</span>We designed 35 different level 2 constructs by using the modular cloning system (MoClo) and transformed these into <i>Chlamydomonas</i> <i>reinhardtii</i>. 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).
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</p>
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</div>
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<div class="figure">
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<img src="https://2019.igem.org/wiki/images/6/6c/T--TU_Kaiserslautern--resultsFigure6.svg"/>
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<p class="caption"><span class="phat">Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS.                                                       
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</span><span class="accent">(a)</span> Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase equipped with secretion signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE) and arylsulfatase (ARS). See Figure 1 for the description of other parts. <span class="accent">(b)</span> UVM4 transformants containing the constructs shown in <span class="accent">(a)</span> were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody.Transformants C12 and A27 introduced in Figures 4 and 5, respectively, served as positive controls. The white arrow points to the MUT-PETase.
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</p>
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<div class="figure">
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<img src="https://2019.igem.org/wiki/images/e/ea/T--TU_Kaiserslautern--resultsFigure11.svg"/>
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<p class="caption"><span class="phat">Quantification of secreted MHETase and MUT-PETase.               
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</span><span class="accent">(a)</span> Transformants generated with constructs C, J, M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Whole-cell extracts of strain B1-TIG-HA for which concentrations of the HA-tagged TIG protein are known are loaded next to the lyophilized supernatants. The black arrow points to MHETase, the white arrows to MUT-PETase. The supernatant of a culture with the UVM4 strain were loaded as negative control. <span class="accent">(b)</span> Maximum cell densities, doubling times, daily growth rates, yields of MHETase and PETase and daily productivity of both combined were calculated for the transformant lines indicated.
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</p>
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</div>
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<h1> The Kaiser Collection </h1>
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<p>
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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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</p>
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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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</p>
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<p>
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Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
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</p>
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<p>
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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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</p>
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<p>
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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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</p>
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Revision as of 10:51, 12 December 2019


L2 spectinomycin resistance + ARS_Mut-PETase

The construct encodes the secretion signal ARS in front of MUT-PETase gene. As selection marker an aadA cassette is used. Constructs encoding the ARS show a higher secretion than constructs encoding an cCa or GLE secretion signal upstream to the MUT-PETase. The MUT-PETase is crucial for the degradation of PET into terephthalic acid and ethylene glycol.

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).

Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase equipped with secretion signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE) and arylsulfatase (ARS). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody.Transformants C12 and A27 introduced in Figures 4 and 5, respectively, served as positive controls. The white arrow points to the MUT-PETase.

Quantification of secreted MHETase and MUT-PETase. (a) Transformants generated with constructs C, J, M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Whole-cell extracts of strain B1-TIG-HA for which concentrations of the HA-tagged TIG protein are known are loaded next to the lyophilized supernatants. The black arrow points to MHETase, the white arrows to MUT-PETase. The supernatant of a culture with the UVM4 strain were loaded as negative control. (b) Maximum cell densities, doubling times, daily growth rates, yields of MHETase and PETase and daily productivity of both combined were calculated for the transformant lines indicated.

The Kaiser Collection

We are proud to present our very own MoClo part collection for C. reinhardtii - the Kaiser collection.

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:

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. 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. Visit our part collection site to get an overview over all parts of the Kaiser Collection

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2401
    Illegal PstI site found at 3491
    Illegal PstI site found at 4464
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2401
    Illegal NheI site found at 2665
    Illegal PstI site found at 3491
    Illegal PstI site found at 4464
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2401
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2401
    Illegal PstI site found at 3491
    Illegal PstI site found at 4464
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2401
    Illegal PstI site found at 3491
    Illegal PstI site found at 4464
    Illegal NgoMIV site found at 1401
    Illegal NgoMIV site found at 1584
    Illegal NgoMIV site found at 1694
    Illegal NgoMIV site found at 3226
    Illegal NgoMIV site found at 3253
    Illegal NgoMIV site found at 4904
  • 1000
    COMPATIBLE WITH RFC[1000]