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
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 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 1104
Illegal PstI site found at 1426
Illegal PstI site found at 1486
Illegal PstI site found at 1958
Illegal PstI site found at 2027
Illegal PstI site found at 2131 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1104
Illegal PstI site found at 1426
Illegal PstI site found at 1486
Illegal PstI site found at 1958
Illegal PstI site found at 2027
Illegal PstI site found at 2131 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1104
Illegal PstI site found at 1426
Illegal PstI site found at 1486
Illegal PstI site found at 1958
Illegal PstI site found at 2027
Illegal PstI site found at 2131 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1104
Illegal PstI site found at 1426
Illegal PstI site found at 1486
Illegal PstI site found at 1958
Illegal PstI site found at 2027
Illegal PstI site found at 2131
Illegal NgoMIV site found at 1348 - 1000COMPATIBLE WITH RFC[1000]
Results
We detected the expression of CYP3A4 with HA-tag (BBa_K4806200) via immunoblotting.
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4 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 spectinomycin-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 CYP3A4 (~ 57 kDa) is visible.
We detected the expression of CYP3A4 with FLAG-tag (BBa_K4806201) via immunoblotting.
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the FLAG-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4 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 spectinomycin-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 CYP3A4 (~ 57 kDa) is visible.
We detected the expression of CYP3A4 tandem together with the POR with HA-tag (BBa_K4806214) via immunoblotting.
(a)Level 2 MoClo construct for expression of the enzyme CYP3A4 tandem together with the POR containing the HA-tag was designed (see Fig.1 for part description)
(b) Picture of resulting western blot. The enzyme CYP3A4/POR 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 CYP3A4 (~ 57 kDa) and the POR (~77 kDa) is visible.
We detected that the construct CYP3A4 with HA-tag (BBa_K4806200) is correctly embedded within the membrane via freeze-thaw assay and immunoblotting.
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag was designed. (b) The UVM4 strain was transformed with the construct in (a). An anti-HA antibody was used to detect the enzyme CYP3A4 (A). For control an anti-Cytf antibody was used to detect the membrane bound proteins and a CGE1-antibody for soluble proteins (B). 30 spectinomycin-resistant transformants were cultivated in TAP-medium and samples taken after. 3 days. For reference whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting. The other samples were treated according to protocol and analyzed also by SDS-PAGE and immunoblotting. The expression of CYP3A4 (~57 kDa) is visible in the pellet (P) and not in the supernatant (S) confirming the membrane intercalation. For reference, the UVM4 strain was used as a negative control.
We were able to detect the expression of CYP3A4 within the pellet, demonstrating that our CYP is correctly embedded within the membrane.
We tried to supertransform the POR with HA-tag (BBa_K4806209) into positive CYP3A4 strains.
(a) Level 2 MoClo constructs for expression of the enzymes CYP3A4 and the POR containing the HA-tag. (b) The CYP3A4 strain was transformed with the POR construct in (a). 30 hygromomycin-resistant transformants were cultivated in TAP-medium and samples taken after 3 days. Whole-cell proteins were extracted and analyzed by SDS-PAGE and immunoblotting using an anti-HA antibody. In the resultant the white arrow marks a cross reaction of antibodies. The expression of CYP3A4 (~57 kDa) is visible. The expression of the POR (~ 77 kDa) is not visible. For reference, the UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) were used as a negative and positive control, respectively.
Sadly we were not able to detect the expression of the POR.
We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806209) by incubating them in the antibiotic Erythromycin
(a) Level 2 MoClo constructs for expression of the enzyme CYP3A4 containing the HA-tag. (b) Positive CYP3A4 transformants were incubated in TAP-medium with different Erythromycin concentration.
Contribution
The * marked parts were not created by us. Our results can be found on the experience page of each part.
The CYurify Collection
The world is at a crossroad. We must decide now how we want to continue living in order to survive. To contribute to this cause, we proudly present our CYPURIFY Collection for Chlamydomonas reinhardtii. The contamination of our water with toxic substances is on the rise, damaging ecosystems and eventually impacting us humans. We see it as our duty to take action.
To accomplish this, we designed 23 level 0, 9 level 1 and 24 level 2 parts for bioremediation of toxic wastewater using Modular Cloning. At heart of this collection are the Cytochrome P450 enzymes. Some of these monooxygenases are already used in synthesis or in medicine. We aimed to take a further step in research by expressing these enzymes in Chlamydomonas for the first time.
Chlamydomonas reinhardtii is the perfect fit for our system as a phototrophic organism with cost-effective and sustainable cultivation. Additionally, this organism is well-studied and easy to transform. We have access to a vast library of preexisting parts, all compatible with Modular Cloning.
Modular Cloning is a cloning method based on the Golden Gate System. What makes it unique is the ability to assemble entire genes in a single reaction. This is made possible by using type IIS restriction enzymes, which cut outside their recognition sequence, effectively removing it after ligation into the target vector. Therefore, the reaction proceeds in a specific direction. The parts are divided into level 0,1 and 2. Level 0 parts are basic components such as promotors, terminators or tags. Level 1 parts are combinations of these level 0 parts, forming transcriptional units. Level 2 parts are combinations of level 1 parts, allowing the expression of multiple genes simultaneously. Level 0 parts are assigned one of 10 positions, with standardized overhangs between them, enabling the exchange of parts between laboratories.
With our collection, we aim to contribute to environmental protection. This collection is infinitely expandable with new CYPs that can degrade other toxic substances. So, what are you waiting for?
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