Difference between revisions of "Part:BBa K4806200"

Revision as of 15:00, 12 October 2023

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

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

Results

We detected the expression of CYP3A4 with HA-tag via immunoblotting.

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.

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.

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.

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.

We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) by incubating UVM4 strains in medium previously incubated by our transformants.

This approach also proved unsuccessful, as all cultures, including the negative control, developed resistance to erythromycin after approximately 1.5 weeks of incubation.

We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via drop-test.

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.

We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via primary antibody.

After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in Chlamydomonas.

We tried to detect the activity of CYP3A4 with mStop (BBa_K4806202) via drop-test.

We dropped all our CYP3A4 transformants with mStop 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.

We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via primary antibody.

After testing several antibody concentrations, we can conclude that the commercial CYP3A4 antibody is not capable of detecting CYP3A4 produced in Chlamydomonas.

We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via HPLC.

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We tried to detect the activity of CYP3A4 with HA-tag (BBa_K4806200) via absorption measurements

Figure 14 demonstrates that the peak at 418 nm, normalized to the total protein content, is elevated in the transformants compared to the wild type, indicating that this new method is valid for screening untagged constructs, but there still a lot more tests to do to verify this method.

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?