Difference between revisions of "Part:BBa K3002029"

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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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<p> <b>
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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>
  
 
 
<div class="figure">
 
<img src="https://2019.igem.org/wiki/images/1/1b/T--TU_Kaiserslautern--resultsFigure4.svg"/>
 
<p class="caption"><span class="phat">Expression of the enzymes MUT-PETase and MHETase in <i>Chlamydomonas</i> <i>reinhardtii</i>.             
 
</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.
 
</p>
 
</div>
 
 
<div class="figure">
 
<img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/>
 
<p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase.                       
 
</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.
 
</p>
 
</div>
 
 
 
<div class="figure">
 
<img src="https://2019.igem.org/wiki/images/5/58/T--TU_Kaiserslautern--resultsFigure9.svg"/>
 
<p class="caption"><span class="phat">Identification of MHETase and MUT-PETase by LC-MS/MS.             
 
</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.
 
</p>
 
</div>
 
 
 
<h1> The Chlamy Yummy Project Collection </h1>
 
<p>
 
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>.
 
</p>
 
<p>
 
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:
 
</p>
 
<p>
 
Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
 
</p>
 
<p>
 
Level 1 parts are combinations of basic parts and usually form functional transcription units.
 
</p>
 
<p>
 
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.
 
</p>
 
<p>
 
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 <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 20:15, 13 December 2019


Wildtype MHETase for Chlamydomonas reinhardtii Part 2 (Phytobrick)

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.

This part was designed to allow synthesis due to a high GC-level. It is only functional in combination with the first part (BBa_K3002005), of the MHETase. Visit the page of the part BBa_K3002037 to gain a deeper understanding of the functionality of the MHETase

Sequence and Features BBa_K3002029 SequenceAndFeatures