Difference between revisions of "Part:BBa K4806100"

 
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<partinfo>BBa_K4806100 short</partinfo>
 
<partinfo>BBa_K4806100 short</partinfo>
  
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<html>
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<style>
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    .bild {max-width: 100% ; height: auto;}
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    .unterschrift {font-size: 11.5px;}
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</style>
  
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<!-- Add more about the biology of this part here
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<p> This level 1 composite part contains the PSAD-promoter (<a href="https://parts.igem.org/Part:BBa_K3002001">BBa_K3002001</a>), the coding sequence of the hygromycin resistance cassette (<a href="https://parts.igem.org/Part:BBa_K4806015">BBa_K4806015</a>)and the PSAD-Terminator (<a href="https://parts.igem.org/Part:BBa_K3002002">BBa_K3002002</a>). This part mediates resistance to spectinomycin. </p>
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===Usage and Biology===
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<br>
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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/por-constructs.png">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  We designed 7 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).
 +
  </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. The POR gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806210">BBa_K4806210</a>)</li>
 +
<li>2. The POR gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806209">BBa_K4806209</a>)</li>
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<li>3. The POR gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806211">BBa_K4806211</a>)</li>
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<li>4. The POR gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806213">BBa_K4806213</a>)</li>
 +
<li>5. The POR gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806212">BBa_K4806212</a>)</li>
 +
<li>6. 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>
 +
<li>7. 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>
 +
</ul>
 +
</p>
 +
<p>
 +
  These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the POR 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 FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or mNeonGreen (<a href=" https://parts.igem.org/Part:BBa_K4806006">BBa_K4806006</a>) for detection or mStop (<a href=" https://parts.igem.org/Part:BBa_K4806009">BBa_K4806009</a>) 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 spectinomycin or hygromycin is already built in the level 2 vector pMBS807 we are using (exept for the tandem construct). 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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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4806100 SequenceAndFeatures</partinfo>
 
  
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<h2>Sequence and Features</h2>
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</html>
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<partinfo>BBa_K4806003 SequenceAndFeatures</partinfo>
  
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<!-- Uncomment this to enable Functional Parameter display
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<partinfo>BBa_K4806003 parameters</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4806100 parameters</partinfo>
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<!-- -->
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<html>
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<h2>Results</h2>
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<p>We detected the expression of the POR with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>) via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-ha.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of the POR with HA-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the POR containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The 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
 +
  </div>
 +
</p>
 +
<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 the POR (~ 77 kDa) is visible.
 +
</p>
 +
<p><br></p>
 +
<p>We detected the expression of the POR with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>) via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-flag.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of the POR with FLAG-tag</b><br>
 +
  (a)Level 2 MoClo construct for expression of the POR containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The 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 FLAG-tagged VIPP1 were used as a negative and positive control, respectively.
 +
  </div>
 +
</p>
 +
<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-FLAG antibody. The expression of the POR (~ 77 kDa) is visible.
 +
</p>
 +
<p><br></p>
 +
<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>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp3a4-tandem-por-wb.png">
 +
  <div class="unterschrift"><b>Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag</b><br>
 +
  (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
 +
  </div>
 +
</p>
 +
<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>
 +
 
 +
<h2>Contribution</h2>
 +
<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
 +
</html>

Revision as of 11:07, 2 October 2023


Hygromycin Resistance for Chlamydomonas reinhardtii (Phytobrick)

This level 1 composite part contains the PSAD-promoter (BBa_K3002001), the coding sequence of the hygromycin resistance cassette (BBa_K4806015)and the PSAD-Terminator (BBa_K3002002). This part mediates resistance to spectinomycin.


Constructs

Fig.1 Construct design
We designed 7 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806210)
  • 2. The POR gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806209)
  • 3. The POR gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806211)
  • 4. The POR gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806213)
  • 5. The POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806212)
  • 6. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
  • 7. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the POR coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) or the PSAD-promotor (BBa_K4806010),either the FLAG-tag (BBa_K4806012), the HA-tag (BBa_K3002017)* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014). The resistance cassette for spectinomycin or hygromycin is already built in the level 2 vector pMBS807 we are using (exept for the tandem construct). 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 611
    Illegal PstI site found at 1871
    Illegal PstI site found at 1931
    Illegal PstI site found at 2590
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1796
    Illegal PstI site found at 611
    Illegal PstI site found at 1871
    Illegal PstI site found at 1931
    Illegal PstI site found at 2590
    Illegal NotI site found at 2236
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 611
    Illegal PstI site found at 1871
    Illegal PstI site found at 1931
    Illegal PstI site found at 2590
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 611
    Illegal PstI site found at 1871
    Illegal PstI site found at 1931
    Illegal PstI site found at 2590
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of the POR with HA-tag (BBa_K4806200) via immunoblotting.

Fig.2 Expression of the POR with HA-tag
(a)Level 2 MoClo construct for expression of the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The 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 the POR (~ 77 kDa) is visible.


We detected the expression of the POR with FLAG-tag (BBa_K4806201) via immunoblotting.

Fig.2 Expression of the POR with FLAG-tag
(a)Level 2 MoClo construct for expression of the POR containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The 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 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 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-FLAG antibody. The expression of the POR (~ 77 kDa) is visible.


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.