Difference between revisions of "Part:BBa K4806100"
Line 13: Line 13:
 
<h2>Constructs</h2>
 
<h2>Constructs</h2>
 
<p>
 
<p>
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/por-constructs.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-1/tandems-construct.png">
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
−
   We designed 7 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).
+
   We designed 2 level 2 constructs containing the hygromycin resistance cassette using the modular cloning system (MoClo).
 
   </div>  
 
   </div>  
 
</p>
 
</p>
Line 22: Line 22:
 
     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 with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806210">BBa_K4806210</a>)</li>
+
<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>
−
<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>
+
<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>
−
<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>
+
−
<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>
 
</ul>
 
</p>
 
</p>
Line 38: Line 33:
 
<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
 
</html>
 
</html>
−
<partinfo>BBa_K4806003 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4806100 SequenceAndFeatures</partinfo>
  
−
<partinfo>BBa_K4806003 parameters</partinfo>
+
<partinfo>BBa_K4806100 parameters</partinfo>
  
  
 
<html>
 
<html>
 
<h2>Results</h2>
 
<h2>Results</h2>
−
<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>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>
 
<p>
Line 72: Line 47:
 
   </div>  
 
   </div>  
 
</p>
 
</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>
+
<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>
 
<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>
 
<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>
 
</html>

Revision as of 11:12, 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 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)

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
    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 1270
    Illegal NgoMIV site found at 1452
  • 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.