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.
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.
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.
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.
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.
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.
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.
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:
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)
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
The theoretical results from SnapGene are shown below: p0_PETase-MHETase ( CDS_B3-B5)
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)
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).
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.