Difference between revisions of "Part:BBa K3002110"
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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>), GLE secretion signal (<a href="https://parts.igem.org/Part:BBa_K3002008">BBa_K3002008</a>), SP20 HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a>) and the coding sequence of the mutant PETase (<a href="https://parts.igem.org/Part:BBa_K3002014">BBa_K3002014</a>) plus the MoClo connectors for positions B1-B2 (<a href="https://parts.igem.org/Part:BBa_K3002302">BBa_K3002302</a>), 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>), GLE secretion signal (<a href="https://parts.igem.org/Part:BBa_K3002008">BBa_K3002008</a>), SP20 HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a>) and the coding sequence of the mutant PETase (<a href="https://parts.igem.org/Part:BBa_K3002014">BBa_K3002014</a>) plus the MoClo connectors for positions B1-B2 (<a href="https://parts.igem.org/Part:BBa_K3002302">BBa_K3002302</a>), 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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The Mut-PETase in combination with the secretion signal GLE and the SP20-tag showed secretion by C.reinhardtii. In constructs without the SP20-tag no secretion of the MUT-PETase was detectable. This applies for the MUT-PETase in combination with the MHETase. In comparison to the other two secretion signals in combination with the sp20 tag, the construct with GLE shows a higher yield of secreted proteins.
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<p></p>
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<div class="figure">
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<img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/>
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<p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase.                       
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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 and MHETase equipped with the secretion signals introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. 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 black arrow points to MHETase, the white arrow to MUT-PETase.
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</div><p>
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</p><div class="figure">
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<img src="https://2019.igem.org/wiki/images/0/0a/T--TU_Kaiserslautern--resultsFigure8.svg"/>
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<p class="caption"><span class="phat">The SP20 module increases the efficiency of protein secretion.
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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 and MHETase equipped with the secretion signals introduced in Figure 6. The constructs contain the coding sequence for a conventional 3xHA tag (C, K, L), or the 3xHA tag preceded by a SP20 tag to enhance glycosylation (M, N, O). See Figure 1 for the description of other parts. <span class="accent">(b)</span> UVM4 transformants containing the constructs C, K, L and M, N, O  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. Transformant A27 introduced in Figures 4, served as positive control. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1 (chloroplast ribosomal 50S protein L1). The RPL1 antibody was used to detect contamination from intracellular proteins.
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</div><p>
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</p>
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<div class="figure">
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<img src="https://2019.igem.org/wiki/images/2/24/T--TU_Kaiserslautern--resultsFigure10.svg"/>
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<p class="caption"><span class="phat">Verification of secretion of MHETase and MUT-PETase into the medium.
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</span>Transformants generated with constructs M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant (s) lyophilized and resuspended in 2xSDS buffer. Cell pellets (p) were also resuspended in SDS-buffer. Both fractions were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. The black arrow points to MHETase, the white arrow to MUT-PETase.
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</p>
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</div><p>
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</p><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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<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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</p>
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<p>
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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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</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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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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</p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 14:23, 10 December 2019


Level 1 - Mutant PETase + GLE + SP20HA (Phytobrick)

This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), GLE secretion signal (BBa_K3002008), SP20 HA-tag (BBa_K3002010) and the coding sequence of the mutant PETase (BBa_K3002014) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).


The Mut-PETase in combination with the secretion signal GLE and the SP20-tag showed secretion by C.reinhardtii. In constructs without the SP20-tag no secretion of the MUT-PETase was detectable. This applies for the MUT-PETase in combination with the MHETase. In comparison to the other two secretion signals in combination with the sp20 tag, the construct with GLE shows a higher yield of secreted proteins.

Effect of the SP20 module on the secretion efficiency of MHETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. 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 black arrow points to MHETase, the white arrow to MUT-PETase.

The SP20 module increases the efficiency of protein secretion. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6. The constructs contain the coding sequence for a conventional 3xHA tag (C, K, L), or the 3xHA tag preceded by a SP20 tag to enhance glycosylation (M, N, O). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs C, K, L and M, N, O 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. Transformant A27 introduced in Figures 4, served as positive control. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1 (chloroplast ribosomal 50S protein L1). The RPL1 antibody was used to detect contamination from intracellular proteins.

Verification of secretion of MHETase and MUT-PETase into the medium. Transformants generated with constructs M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant (s) lyophilized and resuspended in 2xSDS buffer. Cell pellets (p) were also resuspended in SDS-buffer. Both fractions were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. The black arrow points to MHETase, the white arrow to 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 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 1087
    Illegal PstI site found at 2060
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1087
    Illegal PstI site found at 2060
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1087
    Illegal PstI site found at 2060
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1087
    Illegal PstI site found at 2060
    Illegal NgoMIV site found at 822
    Illegal NgoMIV site found at 849
    Illegal NgoMIV site found at 2620
  • 1000
    COMPATIBLE WITH RFC[1000]