Signalling

Part:BBa_K4806014

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-14)


CTPPSAD-transit peptide for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the CTPPSAD-transit peptide to the chloroplast (B2). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with the PSAD-promotor (BBa_K4806010) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression of your target protein in the chloroplast (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 CTPPSAD-transit peptide 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 CTPPSAD-transit peptide the constructs contain the PSAD-promotor (BBa_K4806010), 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/pMBS801 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.


The CYurify Collection

The world is at a crossroad. We must decide now how we want to continue living in order to survive. To contribute to this cause, we proudly present our CYPURIFY Collection for Chlamydomonas reinhardtii. The contamination of our water with toxic substances is on the rise, damaging ecosystems and eventually impacting us humans. We see it as our duty to take action.

To accomplish this, we designed 23 level 0, 9 level 1 and 24 level 2 parts for bioremediation of toxic wastewater using Modular Cloning. At heart of this collection are the Cytochrome P450 enzymes. Some of these monooxygenases are already used in synthesis or in medicine. We aimed to take a further step in research by expressing these enzymes in Chlamydomonas for the first time.

Chlamydomonas reinhardtii is the perfect fit for our system as a phototrophic organism with cost-effective and sustainable cultivation. Additionally, this organism is well-studied and easy to transform. We have access to a vast library of preexisting parts, all compatible with Modular Cloning.

Modular Cloning is a cloning method based on the Golden Gate System. What makes it unique is the ability to assemble entire genes in a single reaction. This is made possible by using type IIS restriction enzymes, which cut outside their recognition sequence, effectively removing it after ligation into the target vector. Therefore, the reaction proceeds in a specific direction. The parts are divided into level 0,1 and 2. Level 0 parts are basic components such as promotors, terminators or tags. Level 1 parts are combinations of these level 0 parts, forming transcriptional units. Level 2 parts are combinations of level 1 parts, allowing the expression of multiple genes simultaneously. Level 0 parts are assigned one of 10 positions, with standardized overhangs between them, enabling the exchange of parts between laboratories.

With our collection, we aim to contribute to environmental protection. This collection is infinitely expandable with new CYPs that can degrade other toxic substances. So, what are you waiting for?

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