Difference between revisions of "Part:BBa K4806217"

 
 
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<partinfo>BBa_K4806217 short</partinfo>
 
<partinfo>BBa_K4806217 short</partinfo>
  
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===Usage and Biology===
 
  
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<p>This composite part contains the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), the CTPPSAD-transit peptide (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>), the CYPCamC coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</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 (Bell, Harford-Cross, Wong, 2001).</p>  
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4806217 SequenceAndFeatures</partinfo>
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<h2>Construct</h2>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cypcamc-ctppsad-construct.png">
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  <div class="unterschrift"><b>Fig.1 Construct design</b><br>
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  This construct was designed using the modular cloning system (MoClo).</div>
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</p>
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  <p>The resistance cassette for hygromycin is already built in the level 2 vector pMBS810 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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<h2>Sequence and Features</h2>
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<partinfo>BBa_K4806217 SequenceAndFeatures</partinfo>
  
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===Functional Parameters===
 
 
<partinfo>BBa_K4806217 parameters</partinfo>
 
<partinfo>BBa_K4806217 parameters</partinfo>
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<h2>Results</h2>
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<p>We confirmed that this construct is built correctly via agarose gel electrophoresis.</p>
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<p>
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  <img class="agarose" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cypcamc-ctppsad-agarose.png">
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  <div class="unterschrift"><b>Fig.2 Test digest of CYPCamC level 2 targeted to the chloroplast</b><br>
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We digested this level 2 MoClo part with the restriction enzymes <i>Hind</i>I and <i>BamH</i>I.</div></p>
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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><br></p>
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<p>We tried to detected the expression of CYPCamC targeted to the chloroplast with HA-tag via immunoblotting.</p>
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<p>
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  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/cymc-neu-ctp.png">
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  <div class="unterschrift"><b>Fig.3 Expression of CYPCamC with the CTPPSAD-tranist peptide</b><br>
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  (a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the CTPPSAD-tranist peptide with 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
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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 CYPCamC (~ 47 kDa) is not visible.
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</p>
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<h2>Contribution</h2>
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<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
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<p><br></p>
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<h2>The CYurify Collection</h2>
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<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>
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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>
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<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>
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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>
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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?
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</p>
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</html>

Latest revision as of 14:20, 8 October 2023


CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the PSAD-promotor (BBa_K4806010), the CTPPSAD-transit peptide (BBa_K4806014), the CYPCamC coding sequence (BBa_K4806002), 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 (Bell, Harford-Cross, Wong, 2001).


Construct

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

The resistance cassette for hygromycin is already built in the level 2 vector pMBS810 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 1205
    Illegal PstI site found at 1477
    Illegal PstI site found at 2201
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1205
    Illegal PstI site found at 1477
    Illegal PstI site found at 2201
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1205
    Illegal PstI site found at 1477
    Illegal PstI site found at 2201
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1205
    Illegal PstI site found at 1477
    Illegal PstI site found at 2201
    Illegal NgoMIV site found at 3222
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

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

Fig.2 Test digest of CYPCamC level 2 targeted to the chloroplast
We digested this level 2 MoClo part with the restriction enzymes HindI and BamHI.

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 detected the expression of CYPCamC targeted to the chloroplast with HA-tag via immunoblotting.

Fig.3 Expression of CYPCamC with the CTPPSAD-tranist peptide
(a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the CTPPSAD-tranist peptide with 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 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 CYPCamC (~ 47 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?