Difference between revisions of "Part:BBa K4806013"
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   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/abetasapi-construct.png">
 
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/abetasapi-construct.png">
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
 
   <div class="unterschrift"><b>Fig.1 Construct design</b><br>
−
   We designed 4 level 2 constructs containing the PSAD-promotor using the modular cloning system (MoClo).
+
   We designed 20 level 2 constructs containing the AβSAP(i)-promotor using the modular cloning system (MoClo).
 
   </div>  
 
   </div>  
 
</p>
 
</p>
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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_K4806206">BBa_K4806206</a>)</li>
 
<li>1. CYP2D6 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</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. CYP2D6 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806207">BBa_K4806207</a>)</li>
−
<li>3. CYP9Q3 gene with FLAG-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>)</li>
+
<li>3. CYP9Q3 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806222">BBa_K4806222</a>)</li>
−
<li>4. 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>4. CYP9Q3 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806223">BBa_K4806223</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>5. CYP9Q3 gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806224">BBa_K4806224</a>)</li>
−
<li>6. 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. 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>
−
<li>7. 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>7. 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>
−
<li>8. 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>8. The POR gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806211">BBa_K4806211</a>)</li>
−
<li>9. 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>9. The POR gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806213">BBa_K4806213</a>)</li>
−
<li>10. 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>10xxxxxxxx. 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>11. 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>11xxxxxxxxxxx. 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>12. 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>12. 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>13. 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>13. CYPCamC gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806216">BBa_K4806216</a>)</li>
−
<li>14. 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>14. CYP81A10V7 gene with HA-tag for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806219">BBa_K4806219</a>)</li>
−
<li>15. 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>15. CYP81A10V7 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806220">BBa_K4806220</a>)</li>
−
<li>16. 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>16. CYP81A10V7 gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806221">BBa_K4806221</a>)</li>
−
<li>17. 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>17. 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>418. 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>18. CYP3A4 gene with mStop for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806202">BBa_K4806202</a>)</li>
−
<li>19. 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>19. 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>20. 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>20. CYP3A4 gene with mNeonGreen for <i>Chlamydomonas reinhardtii</i> (Phytobrick) (<a href=" https://parts.igem.org/Part:BBa_K4806204">BBa_K4806204</a>)</li>
 
</ul>
 
</ul>
 
</p>
 
</p>

Revision as of 15:20, 19 September 2023


AβSAP(i)-promotor for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the AβSAP(i)-promotor (A1-B2). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a coding sequence like CYP3A4 (BBa_K4806000) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression of your target protein (Einhaus et al., 2021). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 20 level 2 constructs containing the AβSAP(i)-promotor using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)
  • 2. CYP2D6 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806207)
  • 3. CYP9Q3 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806222)
  • 4. CYP9Q3 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806223)
  • 5. CYP9Q3 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806224)
  • 6. The POR gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806210)
  • 7. The POR gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806209)
  • 8. The POR gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806211)
  • 9. The POR gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806213)
  • 10xxxxxxxx. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
  • 11xxxxxxxxxxx. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
  • 12. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)
  • 13. CYPCamC gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806216)
  • 14. CYP81A10V7 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806219)
  • 15. CYP81A10V7 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806220)
  • 16. CYP81A10V7 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806221)
  • 17. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
  • 18. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806202)
  • 19. CYP3A4 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806200)
  • 20. CYP3A4 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the PSAD-promotor the constructs contain the CTPPSAD transit peptide to the chloroplast (BBa_K4806014), either the POR (BBa_K4806003), CYP2D6 (BBa_K4806001), CYPCamC (BBa_K4806002) or the CYP3A4 coding sequence (BBa_K4806000), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. The resistance cassette for hygromycin, paromomycin or spectinomycin is already built in the level 2 vector pMBS810/pMBS808/pMBS807 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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (BBa_K4806212, BBa_K4806208, BBa_K4806217, BBa_K4806203) via immunoblotting.

Fig.2 Expression of the POR, CYP2D6, CYPCamC and CYP3A4 in the chloroplast
(1a-4a) Level 2 MoClo construct for expression of the enzyme POR, CYP2D6, CYPCamC and CYP3A4 containing the CTPPSAD transit peptide to the chloroplast were designed (see Fig.1 for part description)
(1b-4b) 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 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 (1a-4a). 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-HA antibody. The expression of the POR (~77 kDa), CYP2D6 (~56 kDa) CYPCamC (~ 47 kDa) and CYP3A4 (~57 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.