Part:BBa_K4806003

POR gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the POR (B3-B4). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promoter like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)^*, this level 0 part leads to expression of the POR. To detect the target protein a tag like HA-tag (BBa_K3002017)^* is recommended.

Constructs

Fig.1 Construct design
We designed 7 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).

Here are the links to the built constructs:

• 1. The POR gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806210)
• 2. The POR gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806209)
• 3. The POR gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806211)
• 4. The POR gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806213)
• 5. The POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806212)
• 6. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)
• 7. CYP2D6 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806215)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the POR coding sequence the constructs contain either the AβSAP(i)-promotor (BBa_K4806013) or the PSAD-promotor (BBa_K4806010),either the FLAG-tag (BBa_K4806012), the HA-tag (BBa_K3002017)^* or mNeonGreen (BBa_K4806006) for detection or mStop (BBa_K4806009) and the tRPL23-terminator (BBa_K3002006)^*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014). The resistance cassette for spectinomycin or hygromycin is already built in the level 2 vector pMBS807 we are using (exept for the tandem construct). 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 611
  Illegal PstI site found at 1871
  Illegal PstI site found at 1931
  Illegal PstI site found at 2590
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1796
  Illegal PstI site found at 611
  Illegal PstI site found at 1871
  Illegal PstI site found at 1931
  Illegal PstI site found at 2590
  Illegal NotI site found at 2236
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 611
  Illegal PstI site found at 1871
  Illegal PstI site found at 1931
  Illegal PstI site found at 2590
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 611
  Illegal PstI site found at 1871
  Illegal PstI site found at 1931
  Illegal PstI site found at 2590
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

We detected the expression of the POR with HA-tag (BBa_K4806200) via immunoblotting.

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

We detected the expression of CYP3A4 with FLAG-tag (BBa_K4806201) 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 CYP3A4 (~ 57 kDa) is visible.

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 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-HA antibody. The expression of CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.

mStop, freeze thaw, mneon green, hplc??????????????????

Contribution

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