Difference between revisions of "Part:BBa K4806205"

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<h1>CYP2D6 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick)</h1>
 
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<p>This composite part contains the paromomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101 ">BBa_K4806101</a>), the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the CYP2D6 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), the HA-tag for detection (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)  and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). This level 2 part was built as part of the CYPurify Collection.<br>
 
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The results show no expression of this level 2 part.
 
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<h2>Results</h2>
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__NOTOC__
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<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p>
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<partinfo>BBa_K4806205 short</partinfo>
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<p><img class="bild" src="">
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<div class="unterschrift"><b>Fig.1 Test digest of CYP2D6 level 2 with HA-tag</b><br>
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<!-- Add more about the biology of this part here
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  We digestes this level 2 MoClo part with the restriction enzymes <i>Sac</i>I & <i>Not</i>I.
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===Usage and Biology===
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  </div> </p>
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<p> The test digest in Fig.1 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.
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<span class='h3bb'>Sequence and Features</span>
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<p>We tried to detect the expression of CYP2D6 with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806205">BBa_K4806205</a>) via immuniblotting.<p>
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<partinfo>BBa_K4806205 SequenceAndFeatures</partinfo>
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<p><img class="bild" src="">
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<div class="unterschrift"><b>Fig.2 Expression of CYP2D6 with HA-tag</b><br>
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  (a)Level2 MoClo construct for expression of the enzymes CYP2D6 containing the HA-tag was designed. <br> Picture of resulting blot. The white arrow marks a cross-reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively.  </div> </p>
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<p> The UVM4 strain was transformed with the construct in (a). 30 paromomycin-resistant transformants were cultivated in TAP and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. The expression of CYP2D6 (~ 56 kDa) is not visible.</p>
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===Functional Parameters===
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<partinfo>BBa_K4806205 parameters</partinfo>
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Revision as of 19:32, 14 September 2023


CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)

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Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NotI site found at 1604
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NgoMIV site found at 1741
    Illegal NgoMIV site found at 2090
    Illegal NgoMIV site found at 3431
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]