Part:BBa_K4806217

CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick)

This composite part contains the PSAD-promotor (BBa_K4806010), the CTPPSAD-transit peptide (BBa_K4806014), the CYPCamC coding sequence (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 potential detoxification of specific chemicals (Ohkawa & Inui, 2015).

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 1205
  Illegal PstI site found at 1477
  Illegal PstI site found at 2201
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1205
  Illegal PstI site found at 1477
  Illegal PstI site found at 2201
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1205
  Illegal PstI site found at 1477
  Illegal PstI site found at 2201
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1205
  Illegal PstI site found at 1477
  Illegal PstI site found at 2201
  Illegal NgoMIV site found at 3222
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

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

Fig.2 Test digest of CYPCamC level 2 targeted to the chloroplast
We digested this level 2 MoClo part with the restriction enzymes HindI and BamHI.

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 detected the expression of CYPCamC targeted to the chloroplast with HA-tag via immunoblotting.

Fig.3 Expression of CYPCamC with the CTPPSAD-tranist peptide
(a)Level 2 MoClo construct for expression of the enzyme CYPCamC containing the CTPPSAD-tranist peptide with 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 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 not visible.

Contribution

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