Difference between revisions of "Part:BBa K4806205"

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<partinfo>BBa_K4806205 short</partinfo>
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4806205 SequenceAndFeatures</partinfo>
 
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     .bild {max-width: 100% ; height: auto;}
 
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<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 CYP2D6 (<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 the tRPL23-terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. 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 potential detoxification of specific chemicals (Ohkawa & Inui, 2015).</p>  
 
<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 CYP2D6 (<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 the tRPL23-terminator (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. 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 potential detoxification of specific chemicals (Ohkawa & Inui, 2015).</p>  
 
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<h2>Constructs</h2>
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<h2>Construct</h2>
 
<p>
 
<p>
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig1.png"/">
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-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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   This construct was degisned using the modular cloning system (MoClo).
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   This construct was designed using the modular cloning system (MoClo).</div>
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  </div>  
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</p>
 
</p>
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  <p>The resistance cassette for paromomycin is already built in the level 2 vector pMBS808 we are using. 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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<p><br></p>
 
<p><br></p>
  
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<html>
 
<html>
 
<h2>Results</h2>
 
<h2>Results</h2>
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<p>We detected the expression of CYP2D6 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>) via immunoblotting.</p>
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<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p>
 
<p>
 
<p>
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp2d6-bba-k4806001-fig2.png">
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   <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/2d6-ha.png">
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   <div class="unterschrift"><b>Fig.2 Expression of CYP2D6 with FLAG-tag</b><br>
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  <div class="unterschrift"><b>Fig.2 Test digest of CYP2D6 level 2 with HA-tag</b><br>
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   (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYP2D6 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.
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We digested this level 2 MoClo part with the restriction enzymes <i>Nco</i>I and <i>Sax</i>I.</div></p>
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  </div>  
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<p>The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.</p>
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</p>
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<p>For detection the UVM4 strain was transformed with the construct in (a). 30 paromomycin-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 CYP2D6 (~ 56 kDa) is visible.
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<p><br></p>
 +
 
 +
<p>We tried to detect the expression of CYP2D6 with HA-tag via immunoblotting.</p>
 +
<p>
 +
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cyp2d6-ha-wb.png">
 +
   <div class="unterschrift"><b>Fig.3 Expression of CYP2D6 with HA-tag</b><br>
 +
   (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. 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>
 +
<p>For detection the UVM4 strain was transformed with the construct in (a). 30 paromomycin-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 CYP2D6 (~ 56 kDa) is not visible.
 +
</p><p><br></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 13:28, 8 October 2023


CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP2D6 (BBa_K4806001), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

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

The resistance cassette for paromomycin is already built in the level 2 vector pMBS808 we are using. 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 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NotI site found at 1604
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NgoMIV site found at 1741
    Illegal NgoMIV site found at 2090
    Illegal NgoMIV site found at 3431
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We confirmed that this construct is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYP2D6 level 2 with HA-tag
We digested this level 2 MoClo part with the restriction enzymes NcoI and SaxI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.


We tried to detect the expression of CYP2D6 with HA-tag via immunoblotting.

Fig.3 Expression of CYP2D6 with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. 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 paromomycin-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 CYP2D6 (~ 56 kDa) is not 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?