Difference between revisions of "Part:BBa K3002001:Experience"

 
 
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===Applications of BBa_K3002001===
 
===Applications of BBa_K3002001===
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<h1>Cytosolic Expression
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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 (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>). 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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Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. The PSAD promoter is a reliable regulatory element for the aadA coding sequence and thus allows a selection of transformants.
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===User Reviews===
 
===User Reviews===

Latest revision as of 19:49, 13 December 2019


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Applications of BBa_K3002001

Cytosolic Expression

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 (BBa_K3002200). The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.

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

User Reviews

UNIQ04c171bf94ec48fb-partinfo-00000001-QINU UNIQ04c171bf94ec48fb-partinfo-00000002-QINU