Difference between revisions of "Part:BBa K4806002"
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   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence the constructs contain a hygromycin resistance cassette (<a href=" https://parts.igem.org/Part:BBa_K4806100">BBa_K4806100</a>), either the βSAP(i)-promotor (AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the PAR-promotor (<a href=" https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a>), or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>) and one the mtTP70C transit peptide to the mitochondria (<a href=" https://parts.igem.org/Part:BBa_K4806011">BBa_K4806011</a>)
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   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP2D6 coding sequence the constructs contain either the βSAP(i)-promotor (AβSAP(i) (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>), the PAR-promotor (<a href=" https://parts.igem.org/Part:BBa_K3002010">BBa_K3002010</a>), or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>) and one the mtTP70C transit peptide to the mitochondria (<a href=" https://parts.igem.org/Part:BBa_K4806011">BBa_K4806011</a>). The resistance cassette for hygromycin is already built in the level 2 vector 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).
 
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Revision as of 19:02, 14 September 2023


CYPCamC gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYPCamC (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 HA-tag (BBa_K3002017) is recommended.


Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing CYPCamC using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYPCamC gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806216)
  • 2. CYPCamC gene for expression in the mitochrondria for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806218)
  • 3. CYPCamC tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence the constructs contain either the βSAP(i)-promotor (AβSAP(i) (BBa_K4806013), the PAR-promotor (BBa_K3002010), or the PSAD-promotor (BBa_K4806010), the HA-tag (BBa_K3002017) for detection and the tRPL23-terminator (BBa_K3002006). Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014) and one the mtTP70C transit peptide to the mitochondria (BBa_K4806011). The resistance cassette for hygromycin is already built in the level 2 vector 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
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 281
    Illegal PstI site found at 553
    Illegal PstI site found at 1277
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 281
    Illegal PstI site found at 553
    Illegal PstI site found at 1277
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 281
    Illegal PstI site found at 553
    Illegal PstI site found at 1277
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 281
    Illegal PstI site found at 553
    Illegal PstI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYPCamC with HA-tag (BBa_K4806216) via immunoblotting.

Fig.2 Expression of CYPCamC with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYPCamC 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 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 (a). 30 hygromycin-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-HA antibody. The expression of CYPCamC (~ 47 kDa) is visible.