Difference between revisions of "Part:BBa K3002010"

 
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===Usage and Biology===
 
===Usage and Biology===
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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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 03:59, 22 October 2019


Sp20 HA tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the sequence of the sp20 3xHA-tag (B5) and was built as a part of the Kaiser Collection. Combined with a secretion signal of the Kaiser Collection like BBa_K3002007 (cCA (B2)), BBa_K3002008 (GLE (B2)) or BBa_K3002009 (ARS (B2)), an appropriate promoter (BBa_K3002001 (PSAD promoter (A1-B1)) or BBa_K3002031 (PAR promoter (A1-B1))) and terminator (e.g. BBa_K3002006 (RPL23 terminator)) your target protein is secreted efficiently and can be detected via HA-antibody (Ramos-Martinez, 2017).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]