Coding

Part:BBa_K4806001

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-13)
Revision as of 14:11, 13 September 2023 by Lulang (Talk | contribs)


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 parts 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 they contain a paromomycin resistance cassette (BBa_K4806101), either the βSAP(i)-promotor (AβSAP(i) (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)

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.

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