Difference between revisions of "Part:BBa K4806010"
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<p>This basic part contains the coding sequence of the PSAD-promotor (A1-B1). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a coding sequence like CYP3A4 (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) and a terminator like tRPL23 (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>), this level 0 part leads to expression of your target protein (Einhaus <i>et al</i>., 2021). To detect the target protein a tag like HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>) is recommended. </p>  
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<p>This basic part contains the coding sequence of the PSAD-promotor (A1-B1). This part is codon-optimized for <i>Chlamydomonas reinhardtii</i> and was built as part of the CYPurify Collection. In combination with a coding sequence like CYP3A4 (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>) and a terminator like tRPL23 (<a href="https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>, this level 0 part leads to expression of your target protein (Einhaus <i>et al</i>., 2021). To detect the target protein a tag like HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> is recommended. </p>  
 
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<h2>Constructs</h2>
 
<h2>Constructs</h2>
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   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the PSAD-promotor the constructs contain the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), CYPCamC (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</a>) or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a> for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>). The resistance cassette for hygromycin, paromomycin or spectinomycin 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).
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   These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the PSAD-promotor the constructs contain the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>), either the POR (<a href=" https://parts.igem.org/Part:BBa_K4806003">BBa_K4806003</a>), CYP2D6 (<a href=" https://parts.igem.org/Part:BBa_K4806001">BBa_K4806001</a>), CYPCamC (<a href=" https://parts.igem.org/Part:BBa_K4806002">BBa_K4806002</a>) or the CYP3A4 coding sequence (<a href=" https://parts.igem.org/Part:BBa_K4806000">BBa_K4806000</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> for detection and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. The resistance cassette for hygromycin, paromomycin or spectinomycin 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).
 
</p>
 
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<p>For detection the UVM4 strain was transformed with the construct in (1a-4a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP2D6 (~56 kDa) CYPCamC (~ 47 kDa) and CYP3A4 (~57 kDa) is not visible.  
 
<p>For detection the UVM4 strain was transformed with the construct in (1a-4a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP2D6 (~56 kDa) CYPCamC (~ 47 kDa) and CYP3A4 (~57 kDa) is not visible.  
 
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<h2>Contribution</h2>
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<p>The <sup>*</sup> marked parts were not created by us. Our results can be found on the experience page of each part.</p>
 
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Revision as of 11:11, 18 September 2023


PSAD-promotor for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of the PSAD-promotor (A1-B1). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a coding sequence like CYP3A4 (BBa_K4806000) and a terminator like tRPL23 (BBa_K3002006)*, this level 0 part leads to expression of your target protein (Einhaus et al., 2021). To detect the target protein a tag like HA-tag (BBa_K3002017)* is recommended.


Constructs

Fig.1 Construct design
We designed 4 level 2 constructs containing the PSAD-promotor using the modular cloning system (MoClo).


Here are the links to the built constructs:

  • 1. The POR gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806212)
  • 2. CYP2D6 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806208)
  • 3. CYPCamC gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806217)
  • 4. CYP3A4 gene for expression in the chloroplast for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806203)

These constructs were transformed into Chlamydomonas reinhardtii. Besides the PSAD-promotor the constructs contain the CTPPSAD transit peptide to the chloroplast (BBa_K4806014), either the POR (BBa_K4806003), CYP2D6 (BBa_K4806001), CYPCamC (BBa_K4806002) or the CYP3A4 coding sequence (BBa_K4806000), the HA-tag (BBa_K3002017)* for detection and the tRPL23-terminator (BBa_K3002006)*. The resistance cassette for hygromycin, paromomycin or spectinomycin 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
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

We tried to detected the expression of the POR, CYP2D6, CYPCamC and CYP3A4 targeted to the chloroplast with HA-tag (BBa_K4806212, BBa_K4806208, BBa_K4806217, BBa_K4806203) via immunoblotting.

Fig.2 Expression of the POR, CYP2D6, CYPCamC and CYP3A4 in the chloroplast
(1a-4a) Level 2 MoClo construct for expression of the enzyme POR, CYP2D6, CYPCamC and CYP3A4 containing the CTPPSAD transit peptide to the chloroplast were designed (see Fig.1 for part description)
(1b-4b) Picture of resulting western blots. The enzymes are marked by a black arrow, the white arrow marks cross reactions 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 (1a-4a). 30 antibiotic resistant transformants (depending on the construct) 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), CYP2D6 (~56 kDa) CYPCamC (~ 47 kDa) and CYP3A4 (~57 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.