Difference between revisions of "Part:BBa K4806000"
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig1.png"/">
 
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig1.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 5 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).
+
   We designed 6 level 2 constructs containing CYP3A4 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. CYP2D6 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806205">BBa_K4806205</a>)</li>
+
<li>1. CYP3A4 gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806201">BBa_K4806201</a>)</li>
−
<li>2. CYP2D6 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806207">BBa_K4806207</a>)</li>
+
<li>2. CYP3A4 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806202">BBa_K4806202</a>)</li>
−
<li>3. CYP2D6 gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>)</li>
+
<li>3. CYP3A4 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806200">BBa_K4806200</a>)</li>
−
<li>4. CYP2D6 gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>)</li>
+
<li>4. CYP3A4 gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806204">BBa_K4806204</a>)</li>
−
<li>5. 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>
+
<li>5. CYP3A4 gene for expression in the chloroplast for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>)</li>
 +
<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>
 
</ul>
 
</ul>
 
</p>
 
</p>
 
<p>
 
<p>
−
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 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 HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>) for detection 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 hygromycin or paromomycin 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).
+
   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP3A4 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/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>
 
</p>
  

Revision as of 08:58, 20 September 2023


CYP3A4 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP3A4 (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 6 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 2. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806202)
  • 3. CYP3A4 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806200)
  • 4. CYP3A4 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)
  • 5. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
  • 6. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP3A4 coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) or the PSAD-promotor (BBa_K4806010),either the FLAG-tag (BBa_K4806012), the HA-tag (BBa_K3002017)* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014). The resistance cassette for spectinomycin or hygromycin is already built in the level 2 vector pMBS807/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 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NotI site found at 862
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NgoMIV site found at 999
    Illegal NgoMIV site found at 1348
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting.

Fig.2 Expression of CYP2D6 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description)
(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.

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.

Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.