Composite

Part:BBa_K4806219

Designed by: Luca Langenberg   Group: iGEM23_RPTU-Kaiserslautern   (2023-09-18)
Revision as of 14:09, 18 September 2023 by Lulang (Talk | contribs)


CYP81A10V7 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)


This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYPCamC (BBa_K4806002), 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 expression and potential detoxification of specific chemicals (Bell et al., 2001).


Construct

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

The resistance cassette for hygromycin is already built in the level 2 vector pMBS810 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 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 249
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NotI site found at 1604
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 530
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1098
    Illegal PstI site found at 2454
    Illegal PstI site found at 2887
    Illegal NgoMIV site found at 1741
    Illegal NgoMIV site found at 2090
    Illegal NgoMIV site found at 3431
    Illegal AgeI site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

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

Fig.2 Test digest of CYP2D6 level 2 with HA-tag
We digested this level 2 MoClo part with the restriction enzymes SacI 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 detect the expression of CYP2D6 with HA-tag via immunoblotting.

Fig.3 Expression of CYP2D6 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the 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 paromomycin-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 CYP2D6 (~ 56 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.

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