Difference between revisions of "Part:BBa K3002014"
Line 13: Line 13:
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
<html>
 +
<p>
 +
The MUT-PETase was expressed with the secretion signals cCA, ARS and GLE by C.reinhardtii. The secretion was successful in combination with the SP20 tag and worked best with the secretion signals cCA and ARS. The Mut-PETase is essential for the degradation of PET into MHET and showed activity against PET and BHET.
 +
 +
</p>
 +
</div><p>
 +
<br/>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).
 +
                    <br/></p>
 +
</div>
 +
 +
<p></p><div class="figure">
 +
<img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/>
 +
<p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase.                       
 +
</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.
 +
</p>
 +
</p>
 +
</html>
  
 
<!-- -->
 
<!-- -->

Revision as of 03:58, 22 October 2019


Mutant PETase for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of a mutant version of the PETase (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii. The following mutations were inserted to increase activity: R280A, S238F and W159H (Joo, S. et al., 2018) (Austin, H. et al, 2018). Combined with a promoter and a terminator, this level 0 construct mediates PET degradation ability. As this part contains the introns 1-3 of RBCS2, it perfectly matches the part BBa_K3002027 (pAR promoter A1-B2), resulting in a high expression (Eichler-Stahlberg et al., 2009). To detect or purify the target protein a tag of the Kaiser Collection like BBa_K3002010 (sp20 HA-tag), BBa_K3002017 (HA-tag), BBa_K3002018 (sp20 His-tag), BBa_K3002028 (His-tag) is recommended. A secretion signal (BBa_K3002007 (cCA), BBa_K3002008 (GLE) or BBa_K3002009 (ARS)) can be added, when using the part BBa_K3002003 (pAR promoter (A1-A3)) as a promoter.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 507
    Illegal PstI site found at 1480
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 507
    Illegal PstI site found at 1480
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 507
    Illegal PstI site found at 1480
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 507
    Illegal PstI site found at 1480
    Illegal NgoMIV site found at 242
    Illegal NgoMIV site found at 269
  • 1000
    COMPATIBLE WITH RFC[1000]