Difference between revisions of "Part:BBa K3002000"
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===Usage and Biology===
 
===Usage and Biology===
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Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. For successful expression and transformation, a reliable selection marker is needed. The aadA coding sequence allows the selection of positive transformants on spectinomycin.
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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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<p class="caption"><span class="phat">Analysis of secreted enzymes of transformant N6 transformed with construct AI.                                     
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</span><span class="accent">(b)</span> Clones generated with transformant N6 (Figure 8) and construct L2AI <span class="accent">(a)</span> were grown in TAP medium for four days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformant C12 introduced in Figure 5, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
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Revision as of 03:37, 22 October 2019


Spectinomycin Resistance for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of a spectinomycin resistance (B3-B5) and was built as a part of the Kaiser Collection. Combined with a promoter (e.g. BBa_K3002003 (PAR promoter (A1-A3)), BBa_K3002034 (Tub2 promoter (A1-A3)), or BBa_K3002036 (PSAD promoter (A1-A3))) and a terminator (e.g. BBa_K3002006 (RPL23 terminator)), this level 0 construct mediates resistance to spectinomycin. It is used as a selection marker in Chlamydomonas and is, therefore, a mandatory component of every level 2 construct.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 586
    Illegal NgoMIV site found at 769
    Illegal NgoMIV site found at 879
  • 1000
    COMPATIBLE WITH RFC[1000]