Difference between revisions of "Part:BBa K4806010"
Line 44: Line 44:
 
<p>We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806212">BBa_K4806212</a>,<a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>,<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>,<a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>) via immunoblotting.</p>
 
<p>We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (<a href=" https://parts.igem.org/Part:BBa_K4806212">BBa_K4806212</a>,<a href=" https://parts.igem.org/Part:BBa_K4806208">BBa_K4806208</a>,<a href=" https://parts.igem.org/Part:BBa_K4806217">BBa_K4806217</a>,<a href=" https://parts.igem.org/Part:BBa_K4806203">BBa_K4806203</a>) via immunoblotting.</p>
 
<p>
 
<p>
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806014-ctppsad-fig2-1.png">
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-1.png">
−
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806014-ctppsad-fig2-2.png">
+
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-2.png.png">
−
   <div class="unterschrift"><b>Fig.2 Expression of CYPCamC with HA-tag</b><br>
+
   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-3.png">
−
   (a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the HA-tag was designed (see Fig.1 for part description) <br> (b) Picture of resulting western blot. The enzyme CYPCamC 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 HA-tagged ribosomal chloroplast 50S protein L5 (RPL5) were used as a negative and positive control, respectively.
+
  <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/ctppsad-bba-k4806014-fig2-4.png">
 +
   <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP2D6, CYPCamC and CYP3A4 in the chloroplast</b><br>
 +
   (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) <br> (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.
 
   </div>  
 
   </div>  
 
</p>
 
</p>
−
<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 visible.  
+
<p>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.  
 
</p>
 
</p>
 
</html>
 
</html>

Revision as of 15:53, 13 September 2023


PSAD-promotor for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the PSAD-promotor (A1-B1). 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 4 level 2 parts containing the PSAD-promotor using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806212)
  • 2. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
  • 3. CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)
  • 4. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the PSAD-promotor the constructs either contain the hygromycin (BBa_K4806100), paromomycin (BBa_K4806101) or the spectinomycin resistance cassette (BBa_K3002000), 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).

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.