Difference between revisions of "Part:BBa K3002104"
 
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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>), cCA secretion signal (<a href="https://parts.igem.org/Part:BBa_K3002007">BBa_K3002007</a>), HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) and the coding sequence of the wildtype PETase (<a href="https://parts.igem.org/Part:BBa_K3002004">BBa_K3002004</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>), cCA secretion signal (<a href="https://parts.igem.org/Part:BBa_K3002007">BBa_K3002007</a>), HA-tag (<a href="https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) and the coding sequence of the wildtype PETase (<a href="https://parts.igem.org/Part:BBa_K3002004">BBa_K3002004</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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This part was used in the constructs L2C (<a href="https://parts.igem.org/Part:BBa_K3002202">BBa_K3002202</a>) and L2H (<a href="https://parts.igem.org/Part:BBa_K3002209">BBa_K3002209</a>).
 
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<img src="https://2019.igem.org/wiki/images/b/b8/T--TU_Kaiserslautern--resultsFigure5.svg"/>
 
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<p class="caption"><span class="phat">MUT-PETase destined for secretion gets stuck inside the cell.                             
 
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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 genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. <span class="accent">(b)</span> Seven days old cultures of transformants generated with the construct shown in <span class="accent">(a)</span> were centrifuged and proteins in the culture medium were precipitated by TCA and analysed by immunoblotting using an anti-HA antibody. The black arrow represents MHETase. <span class="accent">(c)</span> Whole-cell proteins of UVM4 cells transformed with construct L2C (<a href="https://parts.igem.org/Part:BBa_K3002202">BBa_K3002202</a>) shown in <span class="accent">(a)</span> were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>) (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. <span class="accent">(d)</span> Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.
 
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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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<h1> The Chlamy Yummy Project Collection </h1>
 
<h1> The Chlamy Yummy Project Collection </h1>

Latest revision as of 00:31, 14 December 2019


Level 1 Wildtype PETase + cCA + HA (Phytobrick)

This composite part contains the PAR-promoter (BBa_K3002027) in combination with the RPL23-Terminator (BBa_K3002006), cCA secretion signal (BBa_K3002007), HA-tag (BBa_K3002017) and the coding sequence of the wildtype PETase (BBa_K3002004) plus the MoClo connectors for positions B1-B2 (BBa_K3002302), B2-B3 (BBa_K3002303), B4-B5 (BBa_K3002304) and B5-B6 (BBa_K3002305).

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 1069
    Illegal PstI site found at 2042
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1069
    Illegal PstI site found at 2042
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1069
    Illegal PstI site found at 2042
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1069
    Illegal PstI site found at 2042
    Illegal NgoMIV site found at 804
    Illegal NgoMIV site found at 831
    Illegal NgoMIV site found at 2482
  • 1000
    COMPATIBLE WITH RFC[1000]