Composite

Part:BBa_K4806100

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-10-02)
Revision as of 12:03, 4 October 2023 by Lulang (Talk | contribs)


Hygromycin Resistance for Chlamydomonas reinhardtii (Phytobrick)

This level 1 composite part contains the PSAD-promoter (BBa_K3002001), the coding sequence of the hygromycin resistance cassette (BBa_K4806015)and the PSAD-terminator (BBa_K3002002). This part mediates resistance to hygromycin.


Constructs

Fig.1 Construct design
We designed 2 level 2 constructs containing the hygromycin resistance cassette using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
  • 2. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the hygromycin resistance cassette the constructs contains the AβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP3A4 (BBa_K4806000) or CYP2D6 coding sequence (BBa_K4806001), the HA-tag (BBa_K3002017)* for detection and and the tRPL23-terminator (BBa_K3002006)*. Further a dummy for F3 (BBa_K4806017) and an endlinker(BBa_K4806016) were used.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1270
    Illegal NgoMIV site found at 1452
  • 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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