Part:BBa_K4806200
CYP3A4 gene with HA-tag for Chlamydomonas reinhardtii (Phytobrick)
This composite part contains the AβSAP(i)-promotor (BBa_K4806013), the coding sequence of CYP3A4 (BBa_K4806000), 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 expression and potential detoxification of specific chemicals (Ohkawa & Inui, 2015).
Construct
This construct was designed using the modular cloning system (MoClo).
The resistance cassette for spectinomycin is already built in the level 2 vector pMBS807 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1846
Illegal PstI site found at 2168
Illegal PstI site found at 2228
Illegal PstI site found at 2700
Illegal PstI site found at 2769
Illegal PstI site found at 2873 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 249
Illegal PstI site found at 1846
Illegal PstI site found at 2168
Illegal PstI site found at 2228
Illegal PstI site found at 2700
Illegal PstI site found at 2769
Illegal PstI site found at 2873 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 530
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1846
Illegal PstI site found at 2168
Illegal PstI site found at 2228
Illegal PstI site found at 2700
Illegal PstI site found at 2769
Illegal PstI site found at 2873 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1846
Illegal PstI site found at 2168
Illegal PstI site found at 2228
Illegal PstI site found at 2700
Illegal PstI site found at 2769
Illegal PstI site found at 2873
Illegal NgoMIV site found at 2090
Illegal NgoMIV site found at 3592
Illegal AgeI site found at 268 - 1000COMPATIBLE WITH RFC[1000]
Results
We detected the expression of CYP3A4 with HA-tag 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 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 detected that this construct 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 detect the activity of this construct by incubating them in the antibiotic Erythromycin.
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag. (b) Positive CYP3A4 transformants
Our initial assumption was that this test yielded a positive result due to the increased slope of the E26 strain and the associated faster grwoth. Unfortunately, this strain also grows faster without erythromycin, which does not provide a conclusive indication of activity. The E2 strain even exhibits greater sensitivity to erythromycin compared to the wildtype UVM4.
8 Survivability Assay xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
We tried to detect the activity of this construct via drop-test.
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing the HA-tag. (b) 10 µl positive CYP3A4 transformants were dropped on TAP-plates as control. (c) 10 µl positive CYP3A4 transformants were dropped on TAP-plates containing 8 mg/l erythromycin. UVM4 strains were used as negative control.
We dropped all our positive CYP3A4 transformants with HA-tag on plates with and without erythromycin. At this erytrhomycin concentration no wildtype should grow, as previously tested. As the incubation time of this test was much longer than the testing, spontaneous mutations occured in the nagtive control. Therefore this experiment does not verify activity of our enzymes.
6 hplc erythromycin xxxxxxxxxxxxxxxxxxxxxxxxxx
7 estradiol geschichte xxxxxxxxxxxxxxxxxxx
8 nanodrop geschichte xxxxxxxxxxxxx
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?
| None |