Composite

Part:BBa_K4806201

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-18)
Revision as of 10:47, 19 September 2023 by Lulang (Talk | contribs)


CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP3A4 (BBa_K4806000), the FLAG-tag (BBa_K4806012) 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 expression and 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 1846
    Illegal PstI site found at 2168
    Illegal PstI site found at 2228
    Illegal PstI site found at 2700
    Illegal PstI site found at 2769
    Illegal PstI site found at 2873
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1846
    Illegal PstI site found at 2168
    Illegal PstI site found at 2228
    Illegal PstI site found at 2700
    Illegal PstI site found at 2769
    Illegal PstI site found at 2873
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1846
    Illegal PstI site found at 2168
    Illegal PstI site found at 2228
    Illegal PstI site found at 2700
    Illegal PstI site found at 2769
    Illegal PstI site found at 2873
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1846
    Illegal PstI site found at 2168
    Illegal PstI site found at 2228
    Illegal PstI site found at 2700
    Illegal PstI site found at 2769
    Illegal PstI site found at 2873
    Illegal NgoMIV site found at 2090
    Illegal NgoMIV site found at 3165
    Illegal NgoMIV site found at 3171
    Illegal NgoMIV site found at 3583
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We detected the expression of CYP3A4 with FLAG-tag via immunoblotting.

Fig.2 Expression of CYP3A4 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4 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 FLAG-tagged VIPP1 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-FLAG antibody. The expression of CYPCamC (~ 57 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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