Part:BBa_K4806006

mNeonGreen-tag for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of mNeonGreen (B5). This part is codon-optimized for Chlamydomonas reinhardtii and was built as part of the CYPurify Collection. In combination with a promotor like AβSAP(i) (BBa_K4806013) and a terminator like tRPL23 (BBa_K3002006)^*, this level 0 part leads to detection of your expressed target protein like CYP3A4 (BBa_K4806000).

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 mNeonGreen for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806213)
• 2. CYP9Q3 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806224)
• 3. CYP3A4 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806204)
• 4. CYP81A10V7 gene with mStop for Chlamydomonas reinhardtii (Phytobrick) (BBa_K4806221)

These constructs were transformed into Chlamydomonas reinhardtii. Besides mNeonGreen the constructs contain the CAβSAP(i)-promotor (BBa_K4806013), either the POR (BBa_K4806003), CYP9Q3 (BBa_K4806004), CYP3A4 (BBa_K4806000) 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
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 327
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 311
  Illegal PstI site found at 718
  Illegal AgeI site found at 656
  
• 1000
  COMPATIBLE WITH RFC[1000]

Results

We confirmed that CYP3A4 with mNeonGreen (BBa_K4806000) is built correctly via agarose gel electrophoresis.

Fig.2 Test digest of CYP3A4 level 2 with mNeonGreen
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 the POR with mNeonGreen (BBa_K4806003) is built correctly via agarose gel electrophoresis.

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

The test digest in Fig.3 was compared to an in-silico digest. Together with the sequencing results we were able to demonstrate that our construct was built correctly.

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We confirmed that CYP81A10V7 with mNeonGreen (BBa_K4806005) is built correctly via agarose gel electrophoresis.

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

The test digest in Fig.4 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 with mNeonGreen (BBa_K4806004) is built correctly via agarose gel electrophoresis.

Fig.5 Test digest of CYP9Q3 level 2 with mNeonGreen
We digested this level 5 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.

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?