iGEM 2024 — Team sorbonne-u-paris

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{% block title %}Notebook{% endblock %}
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{% block title %}Engineering {% endblock %}
{% block lead %}Mutations prediction of MHETase-PETase{% endblock %}
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{% set sidebar\_items = [
{'href': '#Learn', 'label': 'Learning about PET enzymes'},
{'href': '#PETtoPCA', 'label': 'Circular Economy:from PET to VA'},
{'href': '#PCAtoVA', 'label': 'From PCA to VA'},
] %}
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## Learning about PET's enzymes

Currently, the accumulation of PET waste is steadily increasing and poses a growing threat to ecosystems worldwide. It's estimated that naturally occurring microorganisms take hundreds of years to fully degrade PET plastics. Developing engineered microorganisms capable of breaking down PET into vanillic acid (VA) offers a promising solution, as it transforms plastic waste into a valuable molecule used across various industrial sectors. This approach not only provides a sustainable method for dealing with plastic waste but also supports the concept of a circular economy.
Recently, it was discovered that the bacterium Ideonella sakaiensis is capable of degrading PET plastic into its monomeric components: terephthalic acid (TPA) and ethylene glycol (EG). Japanese scientists were the first to make this breakthrough and to identify the enzymes responsible for the transformation. They showed that two major enzymes PETase and MHETase work synergistically to carry out the following reaction:

![](https://static.igem.wiki/teams/5222/wiki-pages/engineering/petmhettpagraph.png)

Figure 1: PET degradation by PETase and MHETase

Additionally, the article Characterization and Engineering of a Two-Enzyme System for Plastics Depolymerization demonstrated that the PETase-MHETase fusion protein has **greater efficiency** in depolymerizing PET into TPA and EG compared to using the two enzymes separately.

## Circular Economy: Transforming PET into Vanillic Acid

Our team aims to contribute to the circular economy by converting PET, not just into by-products, but into a molecule of economic interest. After analysing all the potential molecules that can be produced from PET and conducting a market study, we identified vanillic acid (VA) as an ideal candidate. VA is relatively easy to produce from PET, and more importantly, it holds significant economic value, as it is utilised across various industries.
In order to convert PET into VA, a series of biochemical reactions is required:
PET -> TPA -> PCA -> VA
In the article *Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6*, it was shown that the enzymes encoded by the tphRICIA2IA3IBIA1I gene cluster are essential for transforming TPA into protocatechuic acid (PCA) through the following reactions:

![](https://static.igem.wiki/teams/5222/wiki-pages/engineering/tpatopcagraph.png)

Figure 2: Conversion of TPA to PCA by Comamonas sp. strain E6. The following gene products are involved in the conversion of TPA to PCA: TphA2I and TphA2II, oxygenase large subunit of TPADO; TphA3I and TphA3II, oxygenase small subunit of TPADO; TphA1I and TphA1II, reductase component of TPADO; and TphBI and TphBII, DCD dehydrogenase,Courtesy of Hirofumi Hara

In fact, the essential enzymes for this transformation are TphA1,A2,A3 ( also noted sometimes as TphAa,Ab,Ac) and TphB.

## Transformation of PCA into Vanillic Acid (VA)

In the article *Biological Valorization of Poly(ethylene terephthalate) Monomers for Upcycling Waste PET*, the authors identified a Homo sapiens O-methyltransferase (HsOMT) enzyme as the most efficient protein for converting protocatechuic acid (PCA) into vanillic acid (VA). Ana Rita Brochado et al. also reported that HsOMT plays a crucial role in vanillin production and that overexpressing this enzyme in a strain with model-guided modifications can significantly increase the final product yield.
Essentially, this enzyme catalyses the methylation reaction, where a methyl group is added to the hydroxyl group on the PCA molecule, converting it into vanillic acid.

![](https://static.igem.wiki/teams/5222/wiki-pages/engineering/tpatova.png)

Figure 3: Reaction cascade to produce Vanillic Acid from TPA and PCA, with the main enzymes TphAabc and TphB

## References

1. Sasoh, Mikio & Masai, Eiji & Ishibashi, Satoko & Hara, Hirofumi & Kamimura, Naofumi & Miyauchi, Keisuke & Fukuda, Masao. (2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and environmental microbiology. 72. 1825-32. 10.1128/AEM.72.3.1825-1832.2006.
2. Duan, Shuyan & Zhang, Nan & Chao, Tianzhu & Wu, Yaoyao & Wang, Mengying. (2023). The structural and molecular mechanisms of type II PETases: a mini review. Biotechnology Letters. 45. 1-15. 10.1007/s10529-023-03418-3.

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# Protein Production from Plasmid pCJ190

## Objective

Produce the fusion protein PETase-MHETase, which includes a 12 AA Gly-Ser linker and a 6 His-tag.

## Transformation Procedure

### 1. Preparation

* Thaw a 50 μL aliquot of NEB 10-beta Competent E. coli (stored at -80°C) on ice. Transfer to a microbiological safety cabinet.
* Add a few μL of plasmid (1 μL of purified plasmid or 2-3 μL of Gibson reaction mixture) to the bacterial aliquot. Gently pipette up and down to mix.

### 2. Heat Shock

* Incubate at 42°C for 30 seconds, then place on ice for 3 minutes.
* Transfer to 37°C for 30 minutes.

### 3. Recovery

* Add 500 μL of LB broth to the electroporation cuvette and gently pipette to mix.
* Transfer 500 μL of the cell suspension to a snap-cap tube and incubate on a shaker at 37°C for 30 minutes to 1 hour.

### 4. Plating

* Prepare agar plates containing Ampicillin (stored at 4°C) and incubate at 37°C.
* Plate 50 μL of the cell suspension on an agar plate using an L-shaped spreader.
* Incubate the plates overnight at 37°C.
* The next day, cover the plates with parafilm and store in a 4°C refrigerator.

# Protein Production: Pre-Culture, Inoculation, Lysis and Incubation with Beads

## Pre-Culture

* Pick a single colony from the plate and grow it overnight in an appropriate volume of LB broth at 37°C.

## Inoculation

* Dilute the overnight culture at a 1:100 ratio in LB medium with the appropriate antibiotic (final concentration of antibiotic: 1:1000).
* Grow at 37°C until the OD600 reaches 0.6 (monitor growth by measuring OD600 approximately 2 hours after inoculation).

## Lysis and Incubation with Beads

### 1. Cell Collection

* Collect 1 mL of the cell suspension and measure its OD600.

### 2. Centrifugation

* Centrifuge cultures at 4000 rpm for 20 minutes at 4°C. Store the pellets at -30°C.

### 3. Lysis of Bacteria

* **Preparation of Lysis Buffer (10 mL total):**
  + 9.4 mL PBS (50 mM PB + 150 mM NaCl)
  + 50 μL MgCl₂ (from a 1 M stock)
  + 50 μL DNase (from a 5 mg/mL stock)
  + 100 μL PMSF (from a 100 mM stock in EtOH)
* On ice, gently resuspend the cell pellet in the lysis buffer (5 mL for <1 L culture, 10-20 mL for >1 L).
* Sonicate in 50 mL Falcon tubes for 5 minutes at 20% amplitude (3 seconds on, 1 second off). Successful lysis is indicated by a color change in the solution.

### 4. DNA Digestion

* Incubate on ice for 2 hours on a shaking platform.
* Centrifuge at 9290 g for 1 hour at 4°C and recover the supernatant.

### 5. Ni-NTA Bead Preparation

* Prepare 2 mL of Ni-NTA beads for every liter of culture media. Wash each 1 mL of beads three times with 1 mL of water and then three times with 1 mL of equilibration buffer (centrifuge for 3 minutes at 3000 rpm and 4°C each time).
* Incubate the supernatant with the washed Ni-NTA beads overnight in a cold room with slow, continuous rotation.

# Column Purification Buffers (pH 7.4)

* **Equilibration:** 50 mL PBS solution
* **Wash 1:** 150 mL PBS + 10 mM Imidazole (34 mg)
* **Wash 2:** 50 mL PBS + 20 mM Imidazole (204 mg)
* **Elution:** 50 mL PBS + 0.5 M Imidazole (1702 mg)

## Purification

### 1. Preparation

* Label three 15 mL Falcon tubes: one for flow-through, one for the first wash, and one for the second wash.
* Prepare 12 labeled 1.7 mL Eppendorf tubes.
* Wash the purification column with water and then with 10 mL of equilibration buffer.

### 2. Loading and Washing

* Pack the column with the SN + Ni-NTA (total volume: 10 mL).
* Keep the flow-through.
* Wash the column with 10 mL of Wash 1 buffer and collect this in the corresponding Falcon tube.
* Repeat with 10 mL of Wash 2 buffer, keeping the collected solution.

### 3. Elution

* Elute with 10 mL of elution buffer into the 1.7 mL Eppendorf tubes (fill each tube to about 800 μL).
* Measure protein concentration using a NanoDrop.

# MoClo Assembly Protocol

## Level 0 Plasmid Construction

### Step 1: Preparing the Tubes and Starting the Reaction

The assembly reaction of the DNA fragments is carried out in 20 μL: 14 μL H₂O + plasmids/PCR and 6 μL of the mix below (Excel to calculate necessary volumes of each component is provided as supplementary information to Crozet et al.).

### Step 2: Preparing the Reaction

* **Assembly Reaction Setup:** The assembly of DNA fragments is performed in a total volume of 20 μL, consisting of:
  + 14 μL H₂O
  + Plasmids/PCR products
  + 6 μL of the assembly mix (volumes of each component can be calculated using the provided Excel file).

![](https://static.igem.wiki/teams/5222/wiki-pages/model/screen1.webp)

![](https://static.igem.wiki/teams/5222/wiki-pages/model/screen2.webp)

* **Recipient Plasmid:** We utilize pICH41308 from the MoClo kit as the recipient plasmid.
* **Thermocycler Program:** The assembly reaction is conducted in a thermocycler using a program that alternates between optimal temperatures for digestion and ligation, lasting approximately 2 hours and 15 minutes. The BbsI sites remain intact during this assembly, allowing for multiple cycles of digestion and ligation.
* **Storage:** The MoClo assembly can be stored at 4°C for short-term use or at -20°C for long-term storage.

### Step 3: Selection and Validation of Assemblies

#### Transformation

* 1 tube of 50 μL NEB 10-beta competent E. coli (stored at -80°C) with 3-4 μL of the MoClo assembly.

#### Procedure

* Remove a tube of bacteria from -80°C for each assembly and control, and place on ice.
* Once thawed, add 3-4 μL of the assembly/control to the tube.
* Heat shock the mixture at 42°C for 45 seconds, then immediately return to ice.
* After 2-3 minutes, add 250 μL of sterile LB broth (without antibiotic) and incubate at 37°C with shaking at 500 rpm for a minimum of 20 minutes (maximum 30 minutes to avoid excessive bacterial growth).
* Plate the entire suspension on LB agar plates containing X-Gal and Spectinomycin.
* Once the plates are dry, invert them and incubate at 37°C overnight. Alternatively, plates can be incubated at 30°C for up to 2-3 days (e.g., over the weekend).

### Step 4: Mini Prep and Validation

#### 1. Mini Prep

* Perform a mini prep using a suitable kit to isolate plasmid DNA.

#### 2. Restriction Digest

* Digest the plasmids using restriction enzymes as follows:
  + NEB T4 DNA Ligase Buffer (10x): 1 μL
  + BsaI: 0.2 μL
  + Plasmid DNA: 200 ng
  + H₂O: adjust to a total volume of 10 μL
* Incubate for 30 minutes to 1 hour at 37°C.

#### 3. Gel Preparation

* Prepare a 1% agarose gel.
* Load the entire digestion reaction onto the gel.

#### 4. Visualization

* Run the gel and visualize the bands using a gel imager or reader.

# SDS-PAGE Gel Preparation Protocol

## 1. Prepare Tubes and Stands

Set up and label the necessary tubes and stands.

## 2. ThermoBlock Setup

Turn on the ThermoBlock and set it to 70ºC.

## 3. Add PBS

Calculate and add PBS to the labeled tubes according to your experimental design.

## 4. Add Samples

Introduce samples and reducing buffer to each tube.

## 5. Centrifuge

Briefly centrifuge the tubes for 5 seconds to mix.

## 6. Heat Samples

Place the tubes in the ThermoBlock and heat for 10 minutes.

## 7. Set Up the SDS Mini Gel Tank

Assemble the electrodes and frames for the SDS Mini Gel Tank.

## 8. Prepare Running Buffer

If needed, prepare the running buffer.

## 9. Fill the Tank

Fill the tank to approximately three-quarters full with the running buffer.

## 10. Install the Gel

Carefully remove the 12 or 15 well SDS gel from its packaging, drain any excess buffer, and gently slide the gel into the tank without fully submerging it.

## 11. Remove the Comb

Gently remove the comb from the gel. Wash the gel and the wells with additional running buffer already present in the tank.

## 12. Load Samples

Add your samples and protein ladders into the gel according to the predetermined layout.

## 13. Submerge the Gel

Add more running buffer to gently submerge the gel fully by sliding it down the frame.

## 14. Cover and Start

Cover the tank (with wires connected) and switch on the machine. It should ramp up to 200V, and small bubbles should be visible rising in the gel near the electrode.

## 15. Run the Gel

Allow the gel to run for approximately 30 minutes or until the blue dye front reaches the bottom of the gel.

## 16. Crack the Gel Casing

Using a spatula, gently crack the gel casing and discard it. Cut off the wells just above the blue line.

## 17. Transfer to Tray #1

Move the gel to Tray #1 containing distilled water.

## 18. Wash the Gel

Wash the gel in distilled water for about 5 minutes in Tray #1.

## 19. Prepare Staining Tray (Tray #2)

In a separate tray (Tray #2), add 20 mL of Page Blue stain.

## 20. Microwave Stain

Add the gel to Tray #2 and microwave for approximately 30 seconds (do not boil).

## 21. Shake Staining Tray

Set Tray #2 to shake for 60 minutes.

## 22. Transfer Gel

Gently remove the gel and transfer it back to Tray #1 or to Tray #3 with distilled water.

## 23. Further Washing

Continue shaking and transfer the gel to fresh distilled water every 15 minutes, or leave it overnight after a couple of washes for optimal clarity.

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## Page Results

Initially, our goal was to express the genes required to convert PET plastic into vanillic acid (VA) in Chlamydomonas reinhardtii, with the aim of creating a photosynthetic organism capable of degrading plastic into a product of economic interest, thus contributing to the circular economy.

Subsequently, we had the idea to perform the same transformation in E. coli in parallel, to compare the efficiency of PET degradation and VA production between these two organisms.

Therefore, we began our experiments with the bacteria first. Unfortunately, due to the constraints imposed by the Olympic Games in Paris, our laboratory was closed for an extended summer period, and we were only able to start the experiments in September.

## Plasmid extraction

The PETase-MHETase fusion protein is capable of converting PET plastic into the by-product TPA, which can subsequently be transformed into vanillic acid (VA) through an enzymatic pathway.

To express PETase-MHETase in E. coli, we ordered the pCJ190 plasmid from Addgene.

This plasmid was delivered in the form of a transformed NEB 10 beta strain, which expresses the pCJ190 plasmid in large quantities.

For our experiments, we initially chose the NEB 10 beta strain, but after discussing with PhD students, we decided to switch to the BL21 strain, which is better suited for high-level expression of proteins of interest. This choice required us to first extract and purify the target plasmid. To do this, we performed a MiniPrep using the GeneJET K0502 kit (Thermo Scientific).

As a result (NanoDrop) we obtained a concentration of 37.7 ng/ml with an A260/280 ratio of 1.97 and an A260/230 ratio of 1.76, indicating that the plasmid is quite pure.

![](https://static.igem.wiki/teams/5222/wiki-pages/results/photoresults1.png)

Figure 1: NanoDrop results of MiniPrep for purification of the pCJ190 plasmid, blank used is Elution buffer of the GeneJET K0502 kit (Thermo Scientific)

To ensure that the purified plasmid corresponds to the target plasmid, we sent 10 µL of the purified solution to NanoPore for sequencing. The results are shown below.

![](https://static.igem.wiki/teams/5222/wiki-pages/results/results3plasmid.png)

![](https://static.igem.wiki/teams/5222/wiki-pages/results/results2.png)

The purified plasmid corresponds perfectly to the pCJ190 plasmid and can therefore be used for the subsequent experiments.

## Transformation of BL21

For the transformation of the BL21 strain with the pCJ190 plasmid, we followed the protocol described in the lab notebook. Indeed, the pCJ190 plasmid, in addition to containing the PETase-MHETase sequence, also carries the ampicillin resistance gene, allowing for the selection of transformed bacteria. We expected that the transformed bacteria would acquire the pCJ190 plasmid along with the ampicillin resistance gene, enabling them to grow on LB agar plates containing ampicillin, while non-transformed bacteria lacking the pCJ190 plasmid would not survive.

After a few unsuccessful attempts, we successfully adapted the heat shock transformation protocol by considering all necessary details (described in detail in the Notebook) and eventually, we obtained the transformed BL21 strains.

![](https://static.igem.wiki/teams/5222/results/fig2a.webp)

![](https://static.igem.wiki/teams/5222/results/fig2b.webp)

Figure 2: A [Up] The photo of the Petri dish containing LB agar with 100 µg/mL of ampicillin after spreading the transformed BL21 strain and incubating overnight at 37°C. We can see some bacterial colonies, which proves the success of the transformation. B [Down] Negative control (BL21 not transformed with pCJ190 + LB Agar with ampicillin 100 ug/mL)

## Liquid culture and induction

After successfully obtaining single colonies, we started a liquid culture of transformed BL21 to later proceed with protein extraction. Once the liquid culture was ready, we induced protein expression using IPTG. The PETase-MHETase gene encoded in the pCJ190 plasmid contains the T7 promoter, which is under the control of the lac operator. This system, T7-lac operator-PETase-MHETase gene, mimics the action of the lactose operon. Without the addition of IPTG (a lactose analogue), the T7 promoter is inhibited by the binding of LacI to the lac operator (LacI is also encoded in pCJ190). Once IPTG is added, the LacI inhibition is removed, and the T7 promoter becomes active, leading to the induction of transcription and thus the expression of the protein of interest.

After IPTG induction, the liquid cultures were left in the incubator at 37°C overnight to ensure bacterial growth. The following day, the OD600 of each culture was measured to check the state of the bacteria and determine if they had grown sufficiently.

The results are shown below.

![](https://static.igem.wiki/teams/5222/results/fig3.webp)

Figure 3: Results of induced bacterial growth after overnight incubation at 37°C

Following overnight incubation of the IPTG-induced BL21 cultures at 37°C, we observed significant bacterial growth. The OD600 measurements showed high values, confirming that the bacteria proliferated well after induction and are ready for the next steps, such as protein extraction.

## Verification of the presence of protein

To verify the presence of our protein, we performed an SDS-PAGE gel using Laemmli buffer, as described in the lab notebook.

![](https://static.igem.wiki/teams/5222/wiki-pages/results/resultsladder.png)

Figure 4: Two SDS-PAGE gels after Coomassie blue staining and an overnight wash with diluted water. In each well, 20 µL of samples (bacterial pellets after centrifugation, diluted with water) were loaded, along with 5 µL of Laemmli buffer x5 with DTT. The molecular weight ladder (DNA ladder) is marked in red.

Unfortunately, no protein was detected. The possible problem: the samples deposited are too diluted + we started from a low number of initial cells. Therefore, after discussing with the PhD students and our PIs, we decided to carry out bacterial lysis, protein purification, and then run a new gel in hopes of visualizing our protein.

Since the PETase-MHETase fusion protein has a 6His-tag, it is possible to isolate the protein by performing Ni-NTA affinity chromatography, as histidines have a high affinity for nickel. After lysing the bacteria by sonication, we performed the purification using Ni-NTA affinity chromatography. The results are shown below.

![](https://static.igem.wiki/teams/5222/results/fig5.webp)

Figure 5: Results of purification of the protein of interest by Ni-NTA affinity chromatography. In the end, after elution, we obtained 13 eppendorfs, each containing 0.8 µL of solution with the purified protein. The A260/280 ratio indicates the purity of the protein, and for a pure protein, it should be around 0.57.

As we can see, the protein concentration is not very high. However, we can use some of the fractions to perform an SDS-PAGE gel and confirm the presence of the protein of interest in these fractions.

The SDS-PAGE gel was performed according to the protocol described in the lab notebook, and the results are shown below.

![](https://static.igem.wiki/teams/5222/wiki-pages/results/resultsgelblue.png)

Figure 6: SDS-PAGE gel after purification: IPTG0 - negative control, non-induced bacteria; FT - flow through (without purification); W1 - Wash 1 (20 mM of imidazole); W2 - Wash 2 (40 mM of imidazole); prot 2 - solution from eppendorf 2 after elution (C = 1.2 mg/mL); prot 3 - solution from eppendorf 3 after elution (C = 0.87 mg/mL); prot 4 - solution from eppendorf 4 after elution (C = 0.8 mg/mL); prot 6 - solution from eppendorf 6 after elution (C = 0.77 mg/mL). The protein of interest (~90 kDa) is highlighted with a red box and we can clearly observe the presence of the protein in tube 2 with the highest concentration.

## Future Experiments in a nutshell

Testing the Activity of PETase-MHETase Enzyme Produced in BL21:
In this step, we plan to extract the protein at a higher concentration from the cell lysate and test its enzymatic activity. Specifically, we will mix the solution containing the protein with a very fine sterile PET film and monitor the production of TPA (terephthalic acid) using mass spectrometry. We hope to obtain quantitative data, which will allow us to extract characteristic values to evaluate the efficiency of the protein. Additionally, we consider using other analytical techniques to assess relevant parameters of the protein.

Sequential Transformation of TPA into VA:
TPA to PCA
Once we confirm the enzymatic activity and the conversion of PET into TPA, we can proceed to the next step. This involves converting the TPA molecule into PCA (protocatechuic acid) and finally into VA (vanillic acid).
To achieve this, we will transform our bacteria (which already contains PETase-MHETase and the ampicillin selection gene) with the plasmid:
1 with plasmid L1\_TphAa,Ab,Ac,B\_Assembled with the kanamycin resistance gene (see page Engineering) which will allow the transformation of TPA into PCA
This step appears to be one of the most challenging since the cluster is quite large, which could pose issues during plasmid production and transformation. Once plasmids are ready and extracted (we will use miniprep to purify our plasmids), we will proceed with the transformation of BL21 bacteria that already contain pCJ190. We will use double selection (ampicillin and kanamycin) to select the necessary bacteria that have 2 plasmids integrated.

Confirmation of PCA Presence:
Before moving to the next step, we must first confirm the presence of PCA in the transformed bacteria. To do this, we plan to lyse the bacteria and, using analytical chemistry methods, attempt to confirm the presence of our molecule (purification + mass spectrometry or NMR).

Transformation of PCA into VA:
Finally, once we are certain that the previous transformation of TPA into PCA has successfully occurred, we can proceed to the last step. This step involves converting the PCA molecule into VA. For this, we will transform our bacteria (which already contains PETase-MHETase, the tph gene cluster, the ampicillin selection gene, and the kanamycin selection gene) with the plasmid:
L1\_HsOMT (see Engineering) containing a gene for O-methyltransferase and the selection gene (chloramphenicol resistance).
Lastly, we will perform a triple bacterial selection (ampicillin, kanamycin, and chloramphenicol). We hope to produce VA, and as in the previous steps, we will use analytical chemistry to extract and detect the VA molecule.

## Experiments with Chlamydomonas

For expressing the PETase-MHETase protein in Chlamydomonas, we plan to use the MoClo technique, the simplified scheme of which is shown below: MoClo Diagram.

Construction of Level 0 Plasmid Containing the Gene of Interest:

To construct the level 0 plasmid containing the DNA sequence encoding the protein of interest, we first had to sequence the DNA we want to integrate and choose the appropriate backbone.

DNA Sequence Construction Containing the Protein of Interest:

As a base sequence, we used the PETase-MHETase DNA with a 12AA linker and 6x HisTag, which is encoded in the plasmid pCJ190. Using bioinformatics tools, we optimised the genetic code to make it compatible with Chlamydomonas (which is GC-rich). We intend to insert our protein into positions B3-B5 of the MoClo system. Therefore, we added appropriate fusion sites at the ends. At the 5' end, we included a B3 scar (AATG) along with the BbsI restriction site, and at the 3' end, a B5 scar along with the BbsI restriction site The construct is shown below:

![](https://static.igem.wiki/teams/5222/wiki-pages/results/resultstable.png)

Level 0:

To construct the Level 0 plasmid containing our synthetic DNA insert, we’ll follow the main steps below:

Step 1: Preparing the Tubes and Starting the Reaction

The assembly reaction of the DNA fragments is carried out in 20 µL: 14 µL H₂O + plasmids/PCR and 6 µL of the mix below (excel to calculate necessary volumes of each component is provided as supplementary information to Crozer et al)

![](https://static.igem.wiki/teams/5222/results/illustration4.webp)

![](https://static.igem.wiki/teams/5222/results/illustration5.webp)

As a recipient plasmid we take pICH41308 from the MoCo kit

The reaction is then performed in the thermocycler using the program below, which alternates optimal temperatures for digestion and ligation, and lasts about 2 hours and 15 minutes. Note that the BbsI sites are not reconstituted during the assembly of the fragments, allowing for cycles of digestion/ligation.

The MoClo assembly can be stored at 4°C (for short-term storage) or at -20°C.

Step 2: Selection/Validation of the Assemblies

Transformation of the MoClo assembly into NEB10 bacteria:

Once the MoClo assemblies are completed (assemblies + controls), they must be transformed:

Step 3: Mini Prep Using a Kit

Step 4: Digestion of Plasmids with Restriction Enzymes and Agarose Gel with DNA revelation
L0 results: The theoretical results from SnapGene for each CDS of interest ( PETase-MHETase, tphall, HsOMT) are shown in the page [Engineering](https://2024.igem.wiki/sorbonne-u-paris/engineering)

The theoretical results from SnapGene are shown below:
p0\_PETase-MHETase ( CDS\_B3-B5)

![](https://static.igem.wiki/teams/5222/results/illustration6.webp)

Level 1:

Here, we aim to assemble the following plasmids:
pCM0-011-PAR (Pro + 5’UTR\_A1-B1)
pCM0-049-mVenus (CDS\_B2)
p0\_PETase-MHETase (CDS\_B3-B5)
pCM0-119-TRPL23 (3’UTR + Ter\_B6-C1)
Positions A1, B1, B2, B3-B5, B6-C1 correspond to the MoClo positions (Crozet et al., 2018).

The plasmids:
pCM0-011-PAR (Pro + 5’UTR\_A1-B1)
pCM0-049-mVenus (CDS\_B2)
pCM0-119-TRPL23 (3’UTR + Ter\_B6-C1)
are derived from the MoClo kit.

For the assembly, we proceed in the same manner as for constructing p0\_PETase-MHETase (CDS\_B3-B5), except this time using the BsaI enzyme instead of BbsI.

p1\_PETase-MHETase-mVenus ( CDS\_B2-B5)

![](https://static.igem.wiki/teams/5222/results/illustration7.webp)

We decided to fuse mVenus with our protein of interest, PETase\_MHETase, in order to visualise the enzyme's localization using fluorescence microscopy.
Additionally, we plan to perform a control experiment without the addition of mVenus to ensure that the fusion of mVenus does not affect the enzyme's function.
For this, we will assemble the following:
pCM0-017-PAR (Pro + 5’UTR\_A1-B1)
p0\_PETase-MHETase (CDS\_B3-B5)
pCM0-119-TRPL23 (3’UTR + Ter\_B6-C1)
The theoretical results from SnapGene are shown below: p1\_PETase-MHETase (CDS\_B3-B5).

![](https://static.igem.wiki/teams/5222/results/illustration8.webp)

Level M in Moclo:
At Level M, we assemble plasmids from Level 1 by combining them with a backbone, a resistance gene, and linkers (sequences that connect DNA fragments). The goal of this assembly is to create a functional plasmid for the organism Chlamydomonas, which includes the genes of interest and allows selection of the transformed cells through the resistance gene.
For the assembly, we proceed in the same manner as for Level 0 but with BbsI enzyme this time. Level M results: The theoretical results from SnapGene for each CDS of interest ( PETase-MHETase, tphall, HsOMT) are shown in the page Engineering
In the end, the assembled plasmid will have the following structure:
Resistance gene – PETaseMHETase – tphall – HsOMT – Final linker.
Afterward, we will carry out the transformation, allowing Chlamydomonas to receive a functional set of genes with all the necessary elements for their expression in the organism. We will then select the transformed Chlamydomonas cells, and we aim to detect the presence of VA (vanillic acid) as the final product using the power of analytical chemistry. We also plan to conduct enzymatic tests and compare the efficiency of the bacterial system versus the Chlamydomonas system in degrading PET and producing VA.

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