Difference between revisions of "Part:BBa K4806012"
 
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   <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag</b><br>
 
   <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag</b><br>
 
   (1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description) <br> (1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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.
 
   (1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description) <br> (1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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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<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>
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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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Latest revision as of 12:11, 6 October 2023


FLAG-tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the FLAG-tag (B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to detection of your expressed target protein like CYP3A4 (BBa_K4806000).


Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing the FLAG-tag using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806210)
  • 2. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the FLAG-tag the constructs either contain the POR (BBa_K4806003), the CYP3A4 (BBa_K4806000) or the CYP2D6 coding sequence(BBa_K4806001), or the CYP3A4 coding sequence (BBa_K4806000) and the tRPL23-terminator (BBa_K3002006)*. The resistance cassette for hygromycin, spectinomycin or paromomycin is already built in the level 2 vector pMBS810/pMBS807/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
    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 1
    Illegal NgoMIV site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP3A4 and CYP2D6 with FLAG-tag (BBa_K4806210, BBa_K4806201, BBa_K4806206) via immunoblotting.

Fig.2 Expression of the POR, CYP3A4, CYP2D6 with FLAG-tag
(1a-3a) Level 2 MoClo constructs for expression of the enzyme POR, CYP3A4, CYP2D6 containing the FLAG-tag were designed (see Fig.1 for part description)
(1b-3b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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.

For detection the UVM4 strain was transformed with the construct in (1a-3a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP3A4 (~57 kDa), and CYP2D6 (~56 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?