Difference between revisions of "Part:BBa K4806012"
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     Here are the links to the built constructs:<br>
 
     Here are the links to the built constructs:<br>
 
<ul>
 
<ul>
−
<li>1. 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>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. 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>
+
<li>2. CYP3A4 gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>)</li>
−
<li>3. CYPCamC gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>)</li>
+
<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>
−
<li>4. CYP3A4 gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>)</li>
+
 
</ul>
 
</ul>
 
</p>
 
</p>
 
<p>
 
<p>
−
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the PSAD-promotor the constructs either contain the hygromycin (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), paromomycin (<a href=" https://parts.igem.org/Part:BBa_K4806101">BBa_K4806101</a>) or the spectinomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K3002000">BBa_K3002000</a>), the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), CYPCamC (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</a>) or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a> for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>).
+
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the FLAG-tag the constructs either contain the hygromycin (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), spectinomycin (<a href=" https://parts.igem.org/Part:BBa_K3002000">BBa_K3002000</a>) or the paromocyin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101">BBa_K4806101</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), the CYP3A4 (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) or the CYP2D6 coding sequence(<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>).
 
</p>
 
</p>
  
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
 
</html>
 
</html>
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<partinfo>BBa_K4806010 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4806012 SequenceAndFeatures</partinfo>
  
−
<partinfo>BBa_K4806010 parameters</partinfo>
+
<partinfo>BBa_K4806012 parameters</partinfo>
  
  
 
<html>
 
<html>
 
<h2>Results</h2>
 
<h2>Results</h2>
−
<p>We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806212">BBa_K4806212</a>, <a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>, <a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>, <a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>) via immunoblotting.</p>
+
<p>We tried to detected the expression of the POR, CYP3A4 and CYP2D6 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806210">BBa_K4806210</a>, <a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>, <a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>) via immunoblotting.</p>
 
<p>
 
<p>
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-1.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/flag-bba-k4806012-fig2-1.png">
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-2.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/flag-bba-k4806012-fig2-2.png">
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-3.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/flag-bba-k4806012-fig2-3.png">
−
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-4.png">
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   <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag</b><br>
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   <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP2D6, CYPCamC and CYP3A4 in the chloroplast</b><br>
+
   (1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description) <br> (1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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.
−
   (1a-4a) Level 2 MoClo construct for expression of the enzyme POR, CYP2D6, CYPCamC and CYP3A4 containing the CTPPSAD transit peptide to the chloroplast were designed (see Fig.1 for part description) <br> (1b-4b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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>  
 
   </div>  
 
</p>
 
</p>
−
<p>For detection the UVM4 strain was transformed with the construct in (1a-4a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP2D6 (~56 kDa) CYPCamC (~ 47 kDa) and CYP3A4 (~57 kDa) is not visible.  
+
<p>For detection the UVM4 strain was transformed with the construct in (1a-3a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP3A4 (~57 kDa), and CYP2D6 (~56 kDa) is visible.  
 
</p>
 
</p>
 
</html>
 
</html>

Revision as of 08:25, 14 September 2023


FLAG-tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the FLAG-tag (B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006), this level 0 part leads to detection of your expressed target protein like CYP3A4 (BBa_K4806000).


Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing the FLAG-tag 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. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the FLAG-tag the constructs either contain the hygromycin (BBa_K4806100), spectinomycin (BBa_K3002000) or the paromocyin resistance cassette (BBa_K4806101), either the POR (BBa_K4806003), the CYP3A4 (BBa_K4806000) or the CYP2D6 coding sequence(BBa_K4806001), or the CYP3A4 coding sequence (BBa_K4806000) and the tRPL23-terminator (BBa_K3002006).

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 1
    Illegal NgoMIV site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP3A4 and CYP2D6 with FLAG-tag (BBa_K4806210, BBa_K4806201, BBa_K4806206) via immunoblotting.

Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag
(1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description)
(1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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 (1a-3a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP3A4 (~57 kDa), and CYP2D6 (~56 kDa) is visible.