Difference between revisions of "Part:BBa K3002113:Experience"

 
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The MHETase in combination with the secretion signal cCA showed constant secretion by C.reinhardtii. This applies for the MHETase alone but also for the MHETase in combination with other level 1 constructs of the PETase. Constructs including cCA lead to a high yield of the secreted MHETase.
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<p class="caption"><span class="phat">MUT-PETase destined for secretion gets stuck inside the cell.                             
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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 genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. <span class="accent">(b)</span> Seven days old cultures of transformants generated with the construct shown in <span class="accent">(a)</span> 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. <span class="accent">(c)</span> Whole-cell proteins of UVM4 cells transformed with construct L2C shown in <span class="accent">(a)</span> 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. <span class="accent">(d)</span> Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.
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Revision as of 01:46, 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_K3002113

User Reviews

The MHETase in combination with the secretion signal cCA showed constant secretion by C.reinhardtii. This applies for the MHETase alone but also for the MHETase in combination with other level 1 constructs of the PETase. Constructs including cCA lead to a high yield of the secreted MHETase.

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.

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