Part:BBa_K4806002

CYPCamC gene for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of CYPCamC (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 (Bell, Harford-Cross, Wong, 2001). To detect the target protein a tag like HA-tag (BBa_K3002017)^* is recommended.

Constructs

Fig.1 Construct design
We designed 3 level 2 constructs containing CYPCamC using the modular cloning system (MoClo).

Here are the links to the built constructs:

• 1. CYPCamC gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806216)
• 2. CYPCamC gene for expression in the mitochrondria for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806218)
• 3. CYPCamC tandem for expression together with the POR for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the CYP2D6 coding sequence the constructs either contain the AβSAP(i)-promotor (BBa_K4806013), the PAR-promotor (BBa_K3002010)^*, or the PSAD-promotor (BBa_K4806010), the HA-tag (BBa_K3002017)^* for detection and the tRPL23-terminator (BBa_K3002006)^*. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (BBa_K4806014) and one the mtTP70C transit peptide to the mitochondria (BBa_K4806011). 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 281
  Illegal PstI site found at 553
  Illegal PstI site found at 1277
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 281
  Illegal PstI site found at 553
  Illegal PstI site found at 1277
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 281
  Illegal PstI site found at 553
  Illegal PstI site found at 1277
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 281
  Illegal PstI site found at 553
  Illegal PstI site found at 1277
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

We detected the expression of CYPCamC with HA-tag (BBa_K4806216) via immunoblotting.

Fig.2 Expression of CYPCamC with HA-tag
(a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYPCamC 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 CYPCamC (~ 47 kDa) is visible.

Contribution

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