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/4/42/T--TU_Kaiserslautern--resultsFigure2.svg"/>
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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">Toxicity test with TPA, EG and EtOH.           
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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>Growth of <i>Chlamydomonas</i> strain UVM4 was tested in TAP medium containing 0 mM (control), 5 mM and 10 mM terephtalic acid (TPA), ethylene glycol (EG) and ethanol (EtOH). The pH in the medium containing TPA was adjusted to pH 7 with potassium acetate. <span class="accent">(a)</span> UVM4 cells were inoculated in 20 mL with 2*105 cells/mL. Growth was measured by counting cells for 5 days. Error bars represent standard error of three biological replicates. <span class="accent">(b)</span> Spot test analysis with the same concentrations of EG, EtOH and TPA as used in <span class="accent">(a)</span>. Note that the UVM4 strain lacks a cell wall and that cells die if they are too diluted.
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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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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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Revision as of 03:17, 22 October 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. 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

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