Regulatory

Part:BBa_K4806013

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-12)
Revision as of 11:59, 6 October 2023 by Lulang (Talk | contribs)


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_K4806205)
  • 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)
  • 11. 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, the POR/CYP3A4 tandem, CYPCamC and CYP3A4 with HA-tag (BBa_K4806209, BBa_K4806214, BBa_K4806216, BBa_K4806200) 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.

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