Difference between revisions of "Part:BBa K4806013"

Revision as of 15:05, 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 4 level 2 constructs containing the PSAD-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 FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)
4. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
5. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
6. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
7. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
8. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
9. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
10. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
11. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
12. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
13. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
14. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
15. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
16. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
17. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
418. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
19. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
20. 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 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.

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 <h2>Constructs</h2>
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-   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/aspai-constructe.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>
     We designed 4 level 2 constructs containing the PSAD-promotor using the modular cloning system (MoClo).