Composite

Part:BBa_K4806106

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-10-02)
Revision as of 10:41, 2 October 2023 by Lulang (Talk | contribs)


POR gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)


This level 1 composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of the POR (BBa_K4806003), 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.


Construct

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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal NheI site found at 2538
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
    Illegal NotI site found at 2978
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1353
    Illegal PstI site found at 2613
    Illegal PstI site found at 2673
    Illegal PstI site found at 3332
    Illegal NgoMIV site found at 4261
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.

Fig.2 Expression of CYP3A4 tandem together with the POR with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR 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 CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.


Contribution

The * marked parts were not created by us. Our results can be found on the experience page of each part.

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