Difference between revisions of "Part:BBa K4806000"

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<partinfo>BBa_K4806000 short</partinfo>
 
<partinfo>BBa_K4806000 short</partinfo>
  
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===Usage and Biology===
 
  
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<p>This basic part contains the coding sequence of CYP3A4 (B3-B4). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) and a terminator like tRPL23 (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> is recommended. </p>
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<span class='h3bb'>Sequence and Features</span>
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<br>
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<partinfo>BBa_K4806000 SequenceAndFeatures</partinfo>
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<h2>Constructs</h2>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig1.png"/">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  We designed 5 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).
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  </div>  
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</p>
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<p><br></p>
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<p>
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    Here are the links to the built constructs:<br>
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<ul>
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<li>1. CYP2D6 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806205">BBa_K4806205</a>)</li>
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<li>2. CYP2D6 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806207">BBa_K4806207</a>)</li>
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<li>3. CYP2D6 gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>)</li>
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<li>4. CYP2D6 gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>)</li>
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<li>5. CYP2D6 tandem for expression together with the POR for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806215">BBa_K4806215</a>)</li>
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</ul>
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</p>
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<p>
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  These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence the constructs contain either the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), either the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or 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>. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>). The resistance cassette for hygromycin or paromomycin is already built in the level 2 vector pMBS810 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).
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<h2>Sequence and Features</h2>
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===Functional Parameters===
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<partinfo>BBa_K4806000 parameters</partinfo>
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<partinfo>BBa_K4806001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4806001 parameters</partinfo>
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<html>
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<h2>Results</h2>
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<p>We detected the expression of CYP2D6 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>) via immunoblotting.</p>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp2d6-bba-k4806001-fig2.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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  </div>  
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</p>
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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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</html>

Revision as of 08:40, 20 September 2023


CYP3A4 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP3A4 (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 5 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806205)
  • 2. CYP2D6 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806207)
  • 3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)
  • 4. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
  • 5. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) or the PSAD-promotor (BBa_K4806010), either the HA-tag (BBa_K3002017)* or the FLAG-tag (BBa_K4806012) for detection and the tRPL23-terminator (BBa_K3002006)*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014). The resistance cassette for hygromycin or paromomycin is already built in the level 2 vector pMBS810 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 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NotI site found at 862
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NgoMIV site found at 999
    Illegal NgoMIV site found at 1348
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) 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.