Coding

Part:BBa_K4806001

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-13)


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

Fig.1 Construct design
We designed 5 level 2 constructs 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 the constructs either contain the AβSAP(i)-promotor (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). The resistance cassette for paromomycin is already built in the level 2 vector pMBS808 we are using (exept for the tandem construct). 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
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NotI site found at 862
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 356
    Illegal PstI site found at 1712
    Illegal PstI site found at 2145
    Illegal NgoMIV site found at 999
    Illegal NgoMIV site found at 1348
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting.

Fig.2 Expression of CYP2D6 with FLAG-tag
(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.


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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