Difference between revisions of "Part:BBa K4806001"
| Line 10: | Line 10: | ||
<h2>Constructs</h2> | <h2>Constructs</h2> | ||
<p> | <p> | ||
| − | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/ | + | <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> | ||
We designed 5 level 2 parts containing CYP2D6 using the modular cloning system (MoClo). | We designed 5 level 2 parts containing CYP2D6 using the modular cloning system (MoClo). | ||
| Line 28: | Line 28: | ||
<p> | <p> | ||
These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence they contain an the paromomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101 ">BBa_K4806101</a>), either the βSAP(i)-promotor (AβSAP(i) (<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>) 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>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>) | These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence they contain an the paromomycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806101 ">BBa_K4806101</a>), either the βSAP(i)-promotor (AβSAP(i) (<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>) 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>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>) | ||
| + | </p> | ||
| + | <h2>Results</h2> | ||
| + | <p>We detected the expression of CYP2D6 with FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806206">BBa_K4806206</a>) via immunoblotting</p> | ||
| + | <p> | ||
| + | <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/bba-k4806001-cyp2d6-fig2.png"> | ||
| + | <div class="unterschrift"><b>Fig.2 Expression of CYP2D6 with FLAG-tag</b><br> | ||
| + | (a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description) <br> (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. | ||
| + | </div> | ||
| + | </p> | ||
| + | <p>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. | ||
</p> | </p> | ||
</html> | </html> | ||
Revision as of 11:16, 13 September 2023
CYP2D6 gene for Chlamydomonas reinhardtii (Phytobrick)
This basic part contains the coding sequence of CYP2D6 (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 FLAG-tag (BBa_K4806012) is recommended.
Constructs
We designed 5 level 2 parts containing CYP2D6 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_K4806205)
- 2. CYP2D6 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806207)
- 3. CYP2D6 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806206)
- 4. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
- 5. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)
These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence they contain an the paromomycin resistance cassette (BBa_K4806101), either the βSAP(i)-promotor (AβSAP(i) (BBa_K4806013) or the PSAD-promotor (BBa_K4806010), either the HA-tag (BBa_K3002017) or the FLAG-tag (BBa_K4806012) for detection and the tRPL23-terminator (BBa_K3002006). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014)
Results
We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting
(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.