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| | To detect or purify the target protein a tag of the Kaiser Collection like <a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a> (sp20 HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>(HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002018">BBa_K3002018</a>(sp20 His-tag), <a href="https://parts.igem.org/Part:BBa_K3002028">BBa_K3002028</a> (His-tag) is recommended. | | To detect or purify the target protein a tag of the Kaiser Collection like <a href="https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a> (sp20 HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>(HA-tag), <a href="https://parts.igem.org/Part:BBa_K3002018">BBa_K3002018</a>(sp20 His-tag), <a href="https://parts.igem.org/Part:BBa_K3002028">BBa_K3002028</a> (His-tag) is recommended. |
| | A secretion signal <a href="https://parts.igem.org/Part:BBa_K3002007">BBa_K3002007</a> (cCA), <a href="https://parts.igem.org/Part:BBa_K3002008">BBa_K3002008</a> (GLE) or <a href="https://parts.igem.org/Part:BBa_K3002009">BBa_K3002009</a> (ARS)) can be added, when using the part <a href="https://parts.igem.org/Part:BBa_K3002003">BBa_K3002003</a> (pAR promoter (A1-A3)) as a promoter. | | A secretion signal <a href="https://parts.igem.org/Part:BBa_K3002007">BBa_K3002007</a> (cCA), <a href="https://parts.igem.org/Part:BBa_K3002008">BBa_K3002008</a> (GLE) or <a href="https://parts.igem.org/Part:BBa_K3002009">BBa_K3002009</a> (ARS)) can be added, when using the part <a href="https://parts.igem.org/Part:BBa_K3002003">BBa_K3002003</a> (pAR promoter (A1-A3)) as a promoter. |
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| | + | This part was designed to allow synthesis due to a high GC-level. It is only functional in combination with the first part (<a href="https://parts.igem.org/Part:BBa_K3002005">BBa_K3002005</a>), of the MHETase. Visit the page of the part <a href="https://parts.igem.org/Part:BBa_K3002037">BBa_K3002037</a> to gain a deeper understanding of the functionality of the MHETase</b> </p> |
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| − | <div class="figure">
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| − | <img src="https://2019.igem.org/wiki/images/1/1b/T--TU_Kaiserslautern--resultsFigure4.svg"/>
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| − | <p class="caption"><span class="phat">Expression of the enzymes MUT-PETase and MHETase in <i>Chlamydomonas</i> <i>reinhardtii</i>.
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| − | </span><span class="accent">(a)</span> Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). <span class="accent">(b)</span> The UVM4 strain was transformed with the construct shown in <span class="accent">(a)</span>. 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27. The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.
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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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| − | </p>
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| − | <img src="https://2019.igem.org/wiki/images/5/58/T--TU_Kaiserslautern--resultsFigure9.svg"/>
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| − | <p class="caption"><span class="phat">Identification of MHETase and MUT-PETase by LC-MS/MS.
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| − | </span><span class="accent">(a)</span> Transformants generated with construct L2N <span class="accent">(d)</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. Protein bands corresponding to those detected with the anti-HA antibody in a gel run in parallel and stained with Coomassie brilliant blue were excised, in-gel digested with trypsin and analyzed by LC-MS/MS. Peptides identified by LC-MS/MS for MHETase (green) and MUT-PETase (purple) are indicated. <span class="accent">(b, c)</span> Sequences of MHETase and MUT-PETase with the peptides detected by LC-MS/MS are highlighted in green and purple, respectively.
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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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| − | 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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| − | </p>
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| | <!-- Add more about the biology of this part here | | <!-- Add more about the biology of this part here |
This basic part contains the coding sequence of the wildtype MHETase (B4). This part is codon-optimized for Chlamydomonas reinhardtii. Combined with the first part of the MHETase (BBa_K3002005), a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the second intron of RBCS2, it perfectly matches the part BBa_K3002027(pAR promoter A1-B2) resulting in a high expression (Eichler-Stahlberg, 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.
