Regulatory

Part:BBa_K4806010

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-13)
Revision as of 14:58, 13 September 2023 by Lulang (Talk | contribs)


PSAD-promotor for Chlamydomonas reinhardtii (Phytobrick)

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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]



PSAD-promotor for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the PSAD-promotor (A1-B1). 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 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 parts 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 they contain a hygromycin resistance cassette (BBa_K4806100), 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)

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.

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