Difference between revisions of "Part:BBa K4806000"
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| − | These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP3A4 coding sequence the constructs contain either the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>),either the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or mNeonGreen (<a href=" https://parts.igem.org/Part:BBa_K4806006">BBa_K4806006</a>) for detection or mStop (<a href=" https://parts.igem.org/Part:BBa_K4806009">BBa_K4806009</a>) and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>). The resistance cassette for spectinomycin or hygromycin is already built in the level 2 vector pMBS807 | + | These constructs were transformed into <i>Chlamydomonas reinhardtii</i>. Besides the CYP3A4 coding sequence the constructs contain either the AβSAP(i)-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806013">BBa_K4806013</a>) or the PSAD-promotor (<a href=" https://parts.igem.org/Part:BBa_K4806010">BBa_K4806010</a>),either the FLAG-tag (<a href=" https://parts.igem.org/Part:BBa_K4806012">BBa_K4806012</a>), the HA-tag (<a href=" https://parts.igem.org/Part:BBa_K3002017">BBa_K3002017</a>)<sup>*</sup> or mNeonGreen (<a href=" https://parts.igem.org/Part:BBa_K4806006">BBa_K4806006</a>) for detection or mStop (<a href=" https://parts.igem.org/Part:BBa_K4806009">BBa_K4806009</a>) and the tRPL23-terminator (<a href=" https://parts.igem.org/Part:BBa_K3002006">BBa_K3002006</a>)<sup>*</sup>. Additionally, one construct contains the CTPPSAD transit peptide to the chloroplast (<a href=" https://parts.igem.org/Part:BBa_K4806014">BBa_K4806014</a>). 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). |
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Revision as of 08:59, 20 September 2023
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
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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145 - 12INCOMPATIBLE 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 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 356
Illegal PstI site found at 1712
Illegal PstI site found at 2145 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
Results
We detected the expression of CYP2D6 with FLAG-tag (BBa_K4806206) via immunoblotting.
(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.