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Revision as of 13:25, 8 October 2023


CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the PSAD-promotor (BBa_K4806010), the CTPPSAD-transit peptide (BBa_K4806014), the CYP3A4 coding sequence (BBa_K4806000), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. This level 2 part leads to potential detoxification of specific chemicals (Ohkawa & Inui, 2015).


Construct

Fig.1 Construct design
This construct was designed using the modular cloning system (MoClo).

The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 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 2028
    Illegal PstI site found at 2350
    Illegal PstI site found at 2410
    Illegal PstI site found at 2882
    Illegal PstI site found at 2951
    Illegal PstI site found at 3055
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2028
    Illegal PstI site found at 2350
    Illegal PstI site found at 2410
    Illegal PstI site found at 2882
    Illegal PstI site found at 2951
    Illegal PstI site found at 3055
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 2028
    Illegal PstI site found at 2350
    Illegal PstI site found at 2410
    Illegal PstI site found at 2882
    Illegal PstI site found at 2951
    Illegal PstI site found at 3055
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 2028
    Illegal PstI site found at 2350
    Illegal PstI site found at 2410
    Illegal PstI site found at 2882
    Illegal PstI site found at 2951
    Illegal PstI site found at 3055
    Illegal NgoMIV site found at 2272
    Illegal NgoMIV site found at 3774
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We confirmed that this construct is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYP3A4 level 2 targeted to the chloroplast
We digested this level 2 MoClo part with the restriction enzymes XhoI and NotI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.


We tried to detected the expression of CYP3A4 targeted to the chloroplast with HA-tag via immunoblotting.

Fig.3 Expression of CYP3A4 with the CTPPSAD-tranist peptide
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the CTPPSAD-tranist peptide with HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. 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 spectinomycin-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 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.