Difference between revisions of "Part:BBa K4806013"

Revision as of 11:48, 6 October 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)
10. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
11x. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)
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 AβSAP(i)-promotor the constructs either contain the CYP2D6 (BBa_K4806001), CYP9Q3 (BBa_K4806004), the POR (BBa_K4806003), CYPCamC (BBa_K4806002), CYP81A10V7 (BBa_K4806005) or the CYP3A4 coding sequence (BBa_K4806000), either the HA-tag (BBa_K3002017)^*, the FLAG-tag (BBa_K4806012) or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)^*. The resistance cassette for paromomycin, spectinomycin or hygromycin is already built in the level 2 vector pMBS808/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
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 249
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 530
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 268
1000
COMPATIBLE WITH RFC[1000]

Results

We detected the expression of the POR, CYP3A4 tandem with the POR, CYPCamC and CYP3A4 with HA-tag (BBa_K4806209, BBa_K4806200, BBa_K4806216, BBa_K4806214) via immunoblotting.

Fig.2 Expression of the POR, CYP3A4 tandem with the POR, CYPCamC and CYP3A4 with HA-tag
(1a-4a) Level 2 MoClo construct for expression of the enzyme POR, CYP3A4 tandem with the POR, CYPCamC and CYP3A4 containing the HA-tag 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), CYPCamC (~ 47 kDa) and CYP3A4 (~57 kDa) is visible.

We detected the expression of the POR, CYP2D6 and CYP3A4 with FLAG-tag (BBa_K4806210, BBa_K4806206, BBa_K4806201 via immunoblotting.

Fig.2 Expression of the POR, CYP2D6 and CYP3A4 with FLAG-tag
(1a-3a) Level 2 MoClo construct for expression of the enzyme POR, CYP2D6 and CYP3A4 containing the FLAG-tag were designed (see Fig.1 for part description)
(1b-3b) 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 FLAG-tagged VIPP1 were used as a negative and positive control, respectively.

For detection the UVM4 strain was transformed with the construct in (1a-3a). 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-FLAG antibody. The expression of the POR (~77 kDa), CYP2D6 (~ 56 kDa) and CYP3A4 (~57 kDa) is visible.

Contribution

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

@@ Line 75: / Line 75: @@
    <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/sap-fig3-1.png">
    <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/cyp2d6-flag-wb.png">
-   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level-0/sap-fig3-3.png">
+   <img class="bild" src="https://static.igem.wiki/teams/4806/wiki/registry/level2/3a4-flag-wb-neu.png">
    <div class="unterschrift"><b>Fig.2 Expression of the POR, CYP2D6 and CYP3A4 with FLAG-tag</b><br>
     (1a-3a) Level 2 MoClo construct for expression of the enzyme POR, CYP2D6 and CYP3A4 containing the FLAG-tag were designed (see Fig.1 for part description) <br> (1b-3b) 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 FLAG-tagged VIPP1 were used as a negative and positive control, respectively.