Difference between revisions of "Part:BBa K4806206"

 
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<partinfo>BBa_K4806206 short</partinfo>
 
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4806206 SequenceAndFeatures</partinfo>
 
  
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<p>This composite part contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the coding sequence of CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) for detection and the tRPL23-terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. This level 2 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015).</p>
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<h2>Construct</h2>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp3a4-flag-construct.png">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  This construct was designed using the modular cloning system (MoClo).</div>
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</p>
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  <p>The resistance cassette for paromomycin is already built in the level 2 vector pMBS808 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022). </p>
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K4806206 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
 
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<partinfo>BBa_K4806206 parameters</partinfo>
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<h2>Results</h2>
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<p>We detected the expression of CYP2D6 with FLAG-tag via immunoblotting.</p>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp3a4-flag-wb.png">
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  <div class="unterschrift"><b>Fig.2 Expression of CYP2D6 with FLAG-tag</b><br>
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  (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP2D6 is marked by a black arrow, the white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the FLAG-tagged VIPP1 were used as a negative and positive control, respectively.
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<p>For detection the UVM4 strain was transformed with the construct in (a). 30 paromomycin-resistant transformants were cultivated in TAP medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is visible.
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</p>
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<h2>Contribution</h2>
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<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
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Revision as of 10:57, 19 September 2023


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


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP2D6 (BBa_K4806001), the FLAG-tag (BBa_K4806012) for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).

The resistance cassette for paromomycin is already built in the level 2 vector pMBS808 we are using. The usage of this vector allows the direct assembly of level 0 parts to level 2 constructs, facilitating the cloning time (Niemeyer & Schroda, 2022).


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 3004
    Illegal NgoMIV site found at 3010
    Illegal NgoMIV site found at 3422
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP2D6 with FLAG-tag via immunoblotting.

Fig.2 Expression of CYP2D6 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP2D6 is marked by a black arrow, the white arrow marks a cross reaction of antibodies. For reference, the UVM4 recipient strain and a strain expressing the FLAG-tagged VIPP1 were used as a negative and positive control, respectively.

For detection the UVM4 strain was transformed with the construct in (a). 30 paromomycin-resistant transformants were cultivated in TAP medium and samples were taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is visible.

Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.