Part:BBa_K4806000

CYP3A4 gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYP3A4 (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 and potential detoxification of specific chemicals (Ohkawa & Inui, 2015). To detect the target protein a tag like HA-tag (BBa_K3002017)^* is recommended.

Constructs

Fig.1 Construct design
We designed 6 level 2 constructs containing CYP3A4 using the modular cloning system (MoClo).

Here are the links to the built constructs:

• 1. CYP3A4 gene with FLAG-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806201)
• 2. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806202)
• 3. CYP3A4 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806200)
• 4. CYP3A4 gene with mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)
• 5. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)
• 6. CYP3A4 tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806214)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP3A4 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 356
  Illegal PstI site found at 1712
  Illegal PstI site found at 2145
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 356
  Illegal PstI site found at 1712
  Illegal PstI site found at 2145
  Illegal NotI site found at 862
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 356
  Illegal PstI site found at 1712
  Illegal PstI site found at 2145
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 356
  Illegal PstI site found at 1712
  Illegal PstI site found at 2145
  Illegal NgoMIV site found at 999
  Illegal NgoMIV site found at 1348
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting.

Fig.2 Expression of CYP2D6 with FLAG-tag
(a)Level 2 MoClo construct for expression of the enzyme CYP2D6 containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP2D6 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 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-FLAG antibody. The expression of CYP2D6 (~ 56 kDa) is visible.

Contribution

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