Difference between revisions of "Part:BBa K4806100"
 
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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>
+
<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 hygromycin. </p>
 
<br>
 
<br>
 
<h2>Constructs</h2>
 
<h2>Constructs</h2>
 
<p>
 
<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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   <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>
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   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>
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     Here are the links to the built constructs:<br>
 
     Here are the links to the built constructs:<br>
 
<ul>
 
<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>
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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. 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>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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<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>
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<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>
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<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>
 
<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).
+
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), 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>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> for detection and and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Further a dummy for F3 (<a href=" https://parts.igem.org/Part:BBa_K4806017">BBa_K4806017</a>) and an endlinker (<a href=" https://parts.igem.org/Part:BBa_K4806016">BBa_K4806016</a>) were used.
 
</p>
 
</p>
  
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<h2>Sequence and Features</h2>
 
<h2>Sequence and Features</h2>
 
</html>
 
</html>
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<partinfo>BBa_K4806003 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K4806100 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K4806003 parameters</partinfo>
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<partinfo>BBa_K4806100 parameters</partinfo>
  
  
 
<html>
 
<html>
 
<h2>Results</h2>
 
<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>
 
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  (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>
 
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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 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>
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   </div>  
 
   </div>  
 
</p>
 
</p>
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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>
+
<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>
 +
<p><br></p>
 +
<h2>The CYurify Collection</h2>
 +
<p>The world is at a crossroad. We must decide now how we want to continue living in order to survive. To contribute to this cause, we proudly present our CYPURIFY Collection for <i>Chlamydomonas reinhardtii</i>. The contamination of our water with toxic substances is on the rise, damaging ecosystems and eventually impacting us humans. We see it as our duty to take action.</p><p>
 +
To accomplish this, we designed 23 level 0, 9 level 1 and 24 level 2 parts for bioremediation of toxic wastewater using Modular Cloning. At heart of this collection are the Cytochrome P450 enzymes. Some of these monooxygenases are already used in synthesis or in medicine. We aimed to take a further step in research by expressing these enzymes in <i>Chlamydomonas</i> for the first time. </p><p>
 +
<i>Chlamydomonas reinhardtii</i> is the perfect fit for our system as a phototrophic organism with cost-effective and sustainable cultivation. Additionally, this organism is well-studied and easy to transform. We have access to a vast library of preexisting parts, all compatible with Modular Cloning.</p><p>
 +
Modular Cloning is a cloning method based on the Golden Gate System. What makes it unique is the ability to assemble entire genes in a single reaction. This is made possible by using type IIS restriction enzymes, which cut outside their recognition sequence, effectively removing it after ligation into the target vector. Therefore, the reaction proceeds in a specific direction. The parts are divided into level 0,1 and 2. Level 0 parts are basic components such as promotors, terminators or tags. Level 1 parts are combinations of these level 0 parts, forming transcriptional units. Level 2 parts are combinations of level 1 parts, allowing the expression of multiple genes simultaneously. Level 0 parts are assigned one of 10 positions, with standardized overhangs between them, enabling the exchange of parts between laboratories. </p><p>
 +
With our collection, we aim to contribute to environmental protection. This collection is infinitely expandable with new CYPs that can degrade other toxic substances. So, what are you waiting for?
 +
</p>
 
</html>
 
</html>

Latest revision as of 11:25, 6 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 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)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP3A4 (BBa_K4806000) or the CYP2D6 coding sequence (BBa_K4806001), the HA-tag (BBa_K3002017)* for detection and and the tRPL23-terminator (BBa_K3002006)*. Further a dummy for F3 (BBa_K4806017) and an endlinker (BBa_K4806016) were used.

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.


The CYurify Collection

The world is at a crossroad. We must decide now how we want to continue living in order to survive. To contribute to this cause, we proudly present our CYPURIFY Collection for Chlamydomonas reinhardtii. The contamination of our water with toxic substances is on the rise, damaging ecosystems and eventually impacting us humans. We see it as our duty to take action.

To accomplish this, we designed 23 level 0, 9 level 1 and 24 level 2 parts for bioremediation of toxic wastewater using Modular Cloning. At heart of this collection are the Cytochrome P450 enzymes. Some of these monooxygenases are already used in synthesis or in medicine. We aimed to take a further step in research by expressing these enzymes in Chlamydomonas for the first time.

Chlamydomonas reinhardtii is the perfect fit for our system as a phototrophic organism with cost-effective and sustainable cultivation. Additionally, this organism is well-studied and easy to transform. We have access to a vast library of preexisting parts, all compatible with Modular Cloning.

Modular Cloning is a cloning method based on the Golden Gate System. What makes it unique is the ability to assemble entire genes in a single reaction. This is made possible by using type IIS restriction enzymes, which cut outside their recognition sequence, effectively removing it after ligation into the target vector. Therefore, the reaction proceeds in a specific direction. The parts are divided into level 0,1 and 2. Level 0 parts are basic components such as promotors, terminators or tags. Level 1 parts are combinations of these level 0 parts, forming transcriptional units. Level 2 parts are combinations of level 1 parts, allowing the expression of multiple genes simultaneously. Level 0 parts are assigned one of 10 positions, with standardized overhangs between them, enabling the exchange of parts between laboratories.

With our collection, we aim to contribute to environmental protection. This collection is infinitely expandable with new CYPs that can degrade other toxic substances. So, what are you waiting for?