Part:BBa_K4806009

mStop for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of mStop (B5). 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.

Constructs

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

Here are the links to the built constructs:

• 1. The POR gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806211)
• 2. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806202)
• 3. CYP9Q3 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806223)
• 4. CYP81A10V7 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806220)

These constructs were transformed into Chlamydomonas reinhardtii. Besides mStop the constructs contain the CAβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP3A3 (BBa_K4806000), CYP9Q3 (BBa_K4806004) or the CYP81A10V7 coding sequence (BBa_K4806005) and the tRPL23-terminator (BBa_K3002006)^*. The resistance cassette for hygromycin or spectinomycin is already built in the level 2 vector pMBS810/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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 14
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Results

We confirmed that CYP3A4 with mStop (BBa_K3002202) is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYP3A4 level 2 with mStop
We digested this level 2 MoClo part with the restriction enzymes sapI and NotI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.

We confirmed that the POR with mStop (BBa_K3002211) is built correctly via agarose gel electrophoresis.

Fig.3 Test digest of the POR level 2 with mStop
We digested this level 2 MoClo part with the restriction enzymes NheI and EcoRV.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.

We confirmed that CYP81A10V7 with mStop (BBa_K3002220) is built correctly via agarose gel electrophoresis.

Fig.4 Test digest of CYP81A10V7 level 2 with mStop
We digested this level 2 MoClo part with the restriction enzymes EcoRV, NdeI and XhoI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.

We confirmed that CYP9Q3 (BBa_K3002223) is built correctly via agarose gel electrophoresis.

Fig.5 Test digest of CYP9Q3 level 2 with mStop
We digested this level 2 MoClo part with the restriction enzymes EcoRV, NdeI and XhoI.

The test digest in Fig.2 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.

We performed a PCR to remove the GS-linker between our CYP and the mStop.

Fig.6 PCR of CYP3A4, 9Q3 and 81A10V7 level 2 with mStop-GS

The PCR product was purified and sequenced.

We tried to detect the activity of CYP81A10V7 (BBa_K3002220) with mStop by plating directly on insecticides

Fig.7 Insecticides
(a) Level 2 MoClo construct for expression of the enzyme CYP81A10V7 containing mStop. (b) CYP81A10V7 were directly plated on HMP and Atrazin. UVM4 on HMP and HMP with Atrazin plates were used as negative control. (c) CYP81A10V7 were directly plated on Chlrosulfuron. UVM4 on Chlorsulfuron plates were used as negative control.

Since there is no difference between the nagtive control and the transformants this experiment was negativ. Also the sequencing results were not correct

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

Fig.8 Drop-test
(a) Level 2 MoClo construct for expression of the enzyme CYP3A4 containing mStop. (b) 10 µl CYP3A4 transformants were dropped on TAP-plates containing 8 mg/l erythromycin. UVM4 strains were used as negative control.

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.

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?