Difference between revisions of "Part:BBa K3002112:Experience"
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| + | <p>The MHETase is expressed in the cytosol of C.reinhardtii. This part is essential for the degradation of MHET into its final degradation products TPA and EG. | ||
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| + | <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. | ||
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Latest revision as of 01:59, 22 October 2019
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The MHETase is expressed in the cytosol of C.reinhardtii. This part is essential for the degradation of MHET into its final degradation products TPA and EG.
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.
UNIQ58b2d210e8b6c90c-partinfo-00000001-QINU UNIQ58b2d210e8b6c90c-partinfo-00000002-QINU