Difference between revisions of "Part:BBa K3002014:Experience"

 
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===Applications of BBa_K3002014===
 
===Applications of BBa_K3002014===
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<img src="https://2019.igem.org/wiki/images/c/cb/T--TU_Kaiserslautern--Goldkriteriumhoffentlich.png"/>
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<p class="caption"><span class="phat">Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas</span>
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                (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control. 
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In conclusion, we improved expression and activitiy of the PETase, <a href="https://parts.igem.org/Part:BBa_K1921003">BBa_K1921003</a>, by finely modifying its expression in Chlamydomonas and significantly improving its PET degradation efficiency.
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<br/></p><div class="figure">
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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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<img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/>
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<p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase.                       
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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 and MHETase equipped with the secretion signals introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. 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 black arrow points to MHETase, the white arrow to MUT-PETase.
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</p>
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</div><p>, </p><div class="figure">
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<img src="https://2019.igem.org/wiki/images/0/06/T--TU_Kaiserslautern--resultsFigure18.svg"/>
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<p class="caption"><span class="phat">Measurement of the activity of MUT-PETase from E.coli against PET by reversed-phase HPLC.
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</span><span class="accent">(a)</span> A standard of 1 mM TPA, MHET and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> Hexa-histidine tagged MUT-PETase purified from E.coli SHuffle T7 was incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. <span class="accent">(c)</span> Purified MUT-PETase was denatured for 10 min at 95°C and incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. Detections were performed at 254 nm. The same measurements are displayed, but the scaling of the axis was set to 2000 mAU on the left and to 50 mAU on the right.
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===User Reviews===
 
===User Reviews===

Latest revision as of 03:41, 22 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3002014

Cytosolic expression of PETase (BBa_K1921003) in Chlamydomonas (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequence for HA-tagged PETase (Mutate M) (b) UVM4 transformants containing construct L2AH were grown in TAP medium for four days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW-molecular weight. Transformant J6 served as positive control. Untransformed UVM4 parent strain served as negative control.

In conclusion, we improved expression and activitiy of the PETase, BBa_K1921003, by finely modifying its expression in Chlamydomonas and significantly improving its PET degradation efficiency.

Analysis of the secretion of MUT-PETase with secretion signals cCA, GLE, and ARS. (a) 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. (b) UVM4 transformants containing the constructs shown in (a) 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.

Effect of the SP20 module on the secretion efficiency of MHETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) 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 black arrow points to MHETase, the white arrow to MUT-PETase.

,

Measurement of the activity of MUT-PETase from E.coli against PET by reversed-phase HPLC. (a) A standard of 1 mM TPA, MHET and BHET dissolved in DMSO was measured by HPLC. (b) Hexa-histidine tagged MUT-PETase purified from E.coli SHuffle T7 was incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. (c) Purified MUT-PETase was denatured for 10 min at 95°C and incubated with PET film for 96 h at 25°C. An aliquot of the reaction buffer was analyzed by HPLC. Detections were performed at 254 nm. The same measurements are displayed, but the scaling of the axis was set to 2000 mAU on the left and to 50 mAU on the right.

User Reviews

UNIQ9b224fada5270b76-partinfo-00000001-QINU UNIQ9b224fada5270b76-partinfo-00000002-QINU