Difference between revisions of "Part:BBa K4806214"

 
Line 3: Line 3:
 
<partinfo>BBa_K4806214 short</partinfo>
 
<partinfo>BBa_K4806214 short</partinfo>
  
−
a
 
  
−
<!-- Add more about the biology of this part here
+
<html>
−
===Usage and Biology===
+
<style>
 +
    .bild {max-width: 100% ; height: auto;}
 +
    .unterschrift {font-size: 11.5px;}
 +
</style>
  
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K4806214 SequenceAndFeatures</partinfo>
 
  
 +
<p>This composite part contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the coding sequence of the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>) and CYP3A4 (<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>)<sup>*</sup> for detection and the tRPL23-terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Further this part contains an endlinker (<a href=" https://parts.igem.org/Part:BBa_K4806016">BBa_K4806016</a>) for connection with the vector. This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. This level 2 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015).</p>
 +
<br>
 +
<h2>Construct</h2>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp3a4-tandem-construct.png">
 +
  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
 +
  This construct was designed using the modular cloning system (MoClo).</div>
 +
</p>
 +
 +
<p><br></p>
 +
 +
<h2>Sequence and Features</h2>
 +
</html>
 +
<partinfo>BBa_K4806214 SequenceAndFeatures</partinfo>
  
−
<!-- Uncomment this to enable Functional Parameter display
 
−
===Functional Parameters===
 
 
<partinfo>BBa_K4806214 parameters</partinfo>
 
<partinfo>BBa_K4806214 parameters</partinfo>
−
<!-- -->
+
 
 +
 
 +
<html>
 +
<h2>Results</h2>
 +
<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>
 +
<p><br></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 12:15, 4 October 2023


POR/CYP3A4 tandem with HA-tag for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of the POR (BBa_K4806003) and CYP3A4 (BBa_K4806000), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. Further this part contains an endlinker (BBa_K4806016) for connection with the vector. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3722
    Illegal PstI site found at 4982
    Illegal PstI site found at 5042
    Illegal PstI site found at 5701
    Illegal PstI site found at 8870
    Illegal PstI site found at 9192
    Illegal PstI site found at 9252
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2618
    Illegal NheI site found at 4907
    Illegal NheI site found at 7273
    Illegal PstI site found at 3722
    Illegal PstI site found at 4982
    Illegal PstI site found at 5042
    Illegal PstI site found at 5701
    Illegal PstI site found at 8870
    Illegal PstI site found at 9192
    Illegal PstI site found at 9252
    Illegal NotI site found at 5347
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2899
    Illegal XhoI site found at 7554
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3722
    Illegal PstI site found at 4982
    Illegal PstI site found at 5042
    Illegal PstI site found at 5701
    Illegal PstI site found at 8870
    Illegal PstI site found at 9192
    Illegal PstI site found at 9252
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3722
    Illegal PstI site found at 4982
    Illegal PstI site found at 5042
    Illegal PstI site found at 5701
    Illegal PstI site found at 8870
    Illegal PstI site found at 9192
    Illegal PstI site found at 9252
    Illegal NgoMIV site found at 1270
    Illegal NgoMIV site found at 1452
    Illegal NgoMIV site found at 6630
    Illegal NgoMIV site found at 9114
    Illegal NgoMIV site found at 10618
    Illegal AgeI site found at 2637
    Illegal AgeI site found at 7292
  • 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.