Difference between revisions of "Part:BBa K4806015"

 
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<partinfo>BBa_K4806015 short</partinfo>
 
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<p> This basic part contains the coding sequence of the hygromycin resistance cassette (B3-B5). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a promotor like PSAD (<a href="https://parts.igem.org/Part:BBa_K3002001">BBa_K3002001</a>), and a terminator like  PSAD (<a href="https://parts.igem.org/Part:BBa_K3002002">BBa_K3002002</a>) this level 0 part mediates resistance to hygromycin. </p>
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===Usage and Biology===
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<h2>Constructs</h2>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-1/tandems-construct.png">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  We designed 2 level 2 constructs containing the hygromycin resistance cassette using the modular cloning system (MoClo).
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<p><br></p>
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    Here are the links to the built constructs:<br>
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<li>1. CYP3A4 tandem for expression together with the POR for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806214">BBa_K4806214</a>)</li>
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<li>2. 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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  Part aufzählung fehlt xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
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<span class='h3bb'>Sequence and Features</span>
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<h2>Sequence and Features</h2>
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<partinfo>BBa_K4806015 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
 
<partinfo>BBa_K4806015 parameters</partinfo>
 
<partinfo>BBa_K4806015 parameters</partinfo>
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<h2>Results</h2>
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<p>We detected the expression of CYP3A4 tandem together with the POR with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806214">BBa_K4806214</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/cyp3a4-tandem-por-wb.png">
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  <div class="unterschrift"><b>Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag</b><br>
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  (a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP3A4/POR 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 HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively
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  </div>
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<p>For detection the UVM4 strain was transformed with the construct in (a). 30 hygromycin-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-HA antibody. The expression of CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.</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 11:23, 2 October 2023


Hygromycin resistance for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the hygromycin resistance cassette (B3-B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like PSAD (BBa_K3002001), and a terminator like PSAD (BBa_K3002002) this level 0 part mediates resistance to hygromycin.


Constructs

Fig.1 Construct design
We designed 2 level 2 constructs containing the hygromycin resistance cassette using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
  • 2. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

Part aufzählung fehlt xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

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 455
    Illegal NgoMIV site found at 637
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.

Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR 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 HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively

For detection the UVM4 strain was transformed with the construct in (a). 30 hygromycin-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-HA antibody. The expression of CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.

Contribution

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