Difference between revisions of "Part:BBa K3002002"
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Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD terminator is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.  
 
Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD terminator is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.  
  
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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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<p class="caption"><span class="phat">Analysis of secreted enzymes of transformant N6 transformed with construct AI.                                     
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</span><span class="accent">(b)</span> Clones generated with transformant N6 (Figure 8) and construct L2AI <span class="accent">(a)</span> were grown in TAP medium for four 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 C12 introduced in Figure 5, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
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Revision as of 14:40, 10 December 2019


Terminator.png

PSAD terminator for Chlamydomonas reinhardtii (Phytobrick)

  • This basic part contains the PSAD terminator (B6-C1) for Chlamydomonas reinhardtii and was built as a part of the Kaiser Collection. Combined with a CDS and a promoter, this level 0 construct leads to a high expression of a target protein.


Secondary Structure

File:Mfold-K3002002-1.png

Measurement

  • [http://openwetware.org/wiki/Cconboy:Terminator_Characterization/Results How these parts were measured]

Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD terminator is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.

Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii. (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). (b) The UVM4 strain was transformed with the construct shown in (a). 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.

MUT-PETase destined for secretion gets stuck inside the cell. (a) 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. (b) Seven days old cultures of transformants generated with the construct shown in (a) 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. (c) Whole-cell proteins of UVM4 cells transformed with construct L2C shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. (d) Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.