Part:BBa_K4806205

CYP2D6 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the paromomycin resistance cassette (BBa_K4806101), the AβSAP(i)-promotor (BBa_K4806013), the CYP2D6 coding sequence (BBa_K4806001), the HA-tag for detection (BBa_K3002017) and the tRPL23-terminator (BBa_K3002006). This level 2 part was built as part of the CYPurify Collection.
The results show no expression of this level 2 part.

Results

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

Fig.1 Test digest of CYP2D6 level 2 with HA-tag
We digestes this level 2 MoClo part with the restriction enzymes SacI & NotI.

The test digest in Fig.1 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 (BBa_K4806205) via immuniblotting.

Fig.2 Expression of CYP2D6 with HA-tag
(a)Level2 MoClo construct for expression of the enzymes CYP2D6 containing the HA-tag was designed.
Picture of resulting 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.

The UVM4 strain was transformed with the construct in (a). 30 paromomycin-resistant transformants were cultivated in TAP and samples 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.