Difference between revisions of "Part:BBa K3002010:Experience"

 
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===User Reviews===
 
===User Reviews===
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The SP20 HA tag leads to glycosylation and enhance the secretion of the enzymes MUT-PETase and MHETase significantly. It was crucial to secrete the MUT-PETase. The glycosylation worked very well but might influence the activity of the proteins.
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<br/>To solve this problem, we generated constructs L2H, L2I, and L2J for the expression of
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                    MUT-PETase alone equipped with the secretions signals from carbonic anhydrase (cCA), gamete lytic
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                    enzyme (GLE), and arylsulfatase (ARS), respectively. While a module encoding the cCA signal is
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                    present in the MoClo kit (Crozet et al., 2018), those for GLE and ARS were generated by us.
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                    Arylsulfatase is essential for the mineralization of sulfate by hydrolyzing sulfate esters under
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                    conditions of sulfate deprivation (deHostos et al., 1988). Gamete lytic enzyme is a metalloprotease
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                    that mediates digestion of the cell wall during gametogenesis (Kinoshita et al., 1992). While we
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                    detected no MUT-PETase in the medium when it was equipped with the cCA signal, weak signals were
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                    detected when it contained secretion signals GLE and ARS (Figure 6).
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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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Latest revision as of 04:00, 22 October 2019


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Applications of BBa_K3002010

User Reviews

The SP20 HA tag leads to glycosylation and enhance the secretion of the enzymes MUT-PETase and MHETase significantly. It was crucial to secrete the MUT-PETase. The glycosylation worked very well but might influence the activity of the proteins.


To solve this problem, we generated constructs L2H, L2I, and L2J for the expression of MUT-PETase alone equipped with the secretions signals from carbonic anhydrase (cCA), gamete lytic enzyme (GLE), and arylsulfatase (ARS), respectively. While a module encoding the cCA signal is present in the MoClo kit (Crozet et al., 2018), those for GLE and ARS were generated by us. Arylsulfatase is essential for the mineralization of sulfate by hydrolyzing sulfate esters under conditions of sulfate deprivation (deHostos et al., 1988). Gamete lytic enzyme is a metalloprotease that mediates digestion of the cell wall during gametogenesis (Kinoshita et al., 1992). While we detected no MUT-PETase in the medium when it was equipped with the cCA signal, weak signals were detected when it contained secretion signals GLE and ARS (Figure 6).

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.

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