Introduction
In 2021, the UNILA-LatAm team participated for the first time in the iGEM competition. As a result, we had a significant social and academic impact in both regional and academic environments. As we were the first and only synthetic biology team formed at our university, our participation in the competition brought significant visibility and interest in the area of synthetic biology in the social and academic environment in which we are inserted. Because of this, new personalities and talents were recruited to be part of this journey in the synthetic biology world.
Now, for our second participation, the discovery of new individuals has enabled us to become one team again through new members by developing a new project theme, a theme that required new and different challenges, solutions, and innovations for regional problems, even for our lab limitations. Because of this new approach, our team is the first to work with the microalgae Chlamydomonas reinhardtii at our university.
Learning about the microalgae
Our journey with the Chlamydomonas reinhardtii began in 2022, when we did extensive studying sessions about this microalgae and all its characteristics, learning about it in articles, journals, professionals, and different iGEM teams wikis that had worked with this organism. The curated facts and characteristics we found were documented, and we added information about the strains we worked with in our developed Chlamy Manual, the ManuAlgae, it is available on our contributtion page.
Obtaining the strains and reagents
After a couple of months of intensive study of our Chlamy, we started looking for options to obtain the strains we needed. Obtaining the strains was very difficult since only a few universities in Brazil currently work with this microalgae. As a result of this, only a few of the various universities and professionals that we reached out to responded. We also considered purchasing the strains, but the importing fees for buying them weren’t in our budget.
Once we discussed those options, our only alternative was to contact our advisor João Molino for guidance about this, and he kindly made the contacts of the professionals who worked with Chlamydomonas reinhardtii available. Only then were we able to receive a donation of the strains from Professor Lívia Seno and her student Isac da Silva. To receive the strains donation, our team member Lucas Ovelar traveled from Foz do Iguaçu to São Paulo, where the Chlamy strains CC-400, CC-1690, and CC-1691 were kindly prepared by Isac and his colleagues at UFABC.
Figure 1. CC-400, CC1690, and CC1691 Chlamydomonas reinhardtii donated strains streaked in TAP agar plates.
Our advisor João Molino also helped us in the construction of our Chlamydomonas plasmid design, donating the plasmid we needed for the tests and backbone for our construct – the pJP32PHL7 plasmid.
We also received a kind donation of the pJP32PHL7 plasmid selection antibiotic we needed – Zeocin from Wesley Luzetti, a student from USP.
Experiment Planning
Work Cells
As part of our synthetic biology project, we are working on the cloning and expression of the FAST-PETase enzyme. For this, we have selected Escherichia coli as our main host organism. The choice of E. coli is due to its efficiency and ease of use in the processes of cloning and expression of recombinant proteins.
The first step in our process is the cloning of our plasmid in E. coli. Once we have ensured the correct insertion of the plasmid, we proceed to the expression phase of the FAST-PETase. This enzyme is essential for our project due to its potential properties and applications in the degradation of plastics.
In parallel, we are exploring the use of Chlamydomonas reinhardtii as a model for the expression and secretion of FAST-PETase, interesting to analyze the possibility of applying a system of eukaryotic expression. Our objective is to evaluate the effectiveness of this organism in the production and secretion of the enzyme.
In addition, we are investigating the feasibility of C. reinhardtii as a model for implementation in water treatment plants. Given the ability of this microalgae to grow in aquatic environments and its potential in biotechnology, we believe it could be an excellent candidate for applications in wastewater treatment plants, where FAST-PETase could play a crucial role in the degradation of plastic contaminants.
Glass Beads Transformation
We embarked on a novel journey of transforming Chlamydomonas reinhardtii using the glass beads method. This decision was primarily driven by the constraints we faced at our university, notably the absence of an electroporator. While electroporation is a widely recognized method for cellular transformation, our context compelled us to explore alternative methodologies to achieve our objectives. Glass bead transformation was a pioneering effort within our research group, as it marked the first instance of employing this particular methodology for the transformation of Chlamydomonas reinhardtii in our laboratory.
The glass beads method, while relatively simple in its approach, requires precision and a deep understanding of the microorganism's physiology. Our initial trials focused on optimizing the conditions for successful transformation, considering factors such as bead size, agitation speed, and duration of contact between the beads and the microalgae.
Plastic Degradation
We embarked on a project into plastic degradation. As a proof of concept, we are focusing on the biodegradability of polycaprolactone (PCL) when combined with TAP (Tris-Acetate-Phosphate) medium.
Polycaprolactone, a biodegradable polyester, has garnered attention in environmental research due to its potential to alleviate some of the challenges traditional plastics pose. The TAP medium, known for its nutrient-rich composition, was chosen to provide an optimal environment for expressing the enzymes. See the design page to learn more about our enzymes.
Our experiments involved inoculating Chlamydomonas reinhardtii with our different circuits in PCL plates under controlled conditions, monitoring changes in the physical and chemical structure of the plastic over time. Throughout the study, we expect to observe a noticeable reduction in the weight and structural integrity of the PCL samples. Further microscopic analysis is expected to show signs of surface erosion, indicative of microbial colonization and enzymatic activity breaking down the polymer chains.
This test serves to infer the capacity of microalgae to express hydrolases that degrade polyesters such as polycaprolactone, and insights underscore the potential role of microalgae in addressing the global plastic waste challenge, offering a promising avenue for future research and applications in bioremediation.
In that sense, we planned our experiments in two parts. The first part is focused on the development of the tests in E. coli, and the second part is focused on the familiarization and the development of all our tests in C. reinhardtii.
Part 1: Development stages for E. coli
| Stage | Experiments |
|---|---|
| 1. Preparation and Cloning | Culture medium preparation (solid and liquid) |
| 1. Preparation and Cloning | Reactivation and chemical competence evaluation tests of DH5A cells |
| 1. Preparation and Cloning | E. coli expression plasmid cloning with DH5A cells |
| 1. Preparation and Cloning | E. coli expression plasmid linearization by PCR |
| 2. Expression | Assembly of the FAST-PETase designed constructs with the E. coli expression plasmid linearized vector |
| 2. Expression | Transformation of the DH5A competent cells with assembled plasmid by heat shock |
| 2. Expression | Colony PCR |
| 2. Expression | Plasmid Miniprep |
| 2. Expression | BL21 competent cells transformation by heat shock method |
| 2. Expression | Expression induction with IPTG |
| 3. Degradation | Degradation test with polycaprolactone solid medium (PCL-LB) |
| 3. Degradation | Results evaluation |
Table 1. Experiment Planning for Escherichia coli
Preparation and Cloning
Reactivation and chemical competence evaluation tests of DH5A cells
This step involved the reactivation of chemically competent cells available in our lab. The goal was to linearize the E. coli expression plasmid and facilitate the assembly stage so the overlaps would serve this purpose.
We also tested a new protocol for inducing chemical competence in E. coli cells. It was conducted to evaluate the levels of chemical competence that we can obtain with this method and the possible usage of these cells in the development of our project.
E. coli expression plasmid cloning with DH5A cells
The E. coli expression plasmid is a construct designed by our team in 2021 for the project BioPank. We decided to use the backbone of this plasmid as a vector for our E. coli constructs because of the expression success in using this plasmid.
Figure 2. Expression plasmid from Benchling
E. coli expression plasmid linearization by PCR
We used the platform Benchling to develop all the designs, so we designed primers and overlaps that could complement the E. coli expression vector sequence.
Figure 3. E. coli expression plasmid + FAST-PETase linearization primers
Figure 4. E. coli expression vector + FAST-PETase plasmid
Expression
FAST-PETase and FAST-PETase_Linker_MHETase expression in E. coli
After the linearization, it is time to assemble our expression cassettes. We aimed to assemble our designed parts into the linearized vector and, subsequently, test its expression.
Assembly of the FAST-PETase designed constructs with the E. coli expression plasmid linearized vector
To execute the assembly of our parts, we wanted to use the Gibson Assembly Method and, alternatively, the Hi-Fi assembly method. The choice was made based on the familiarity of some wet lab members with the Gibson Assembly and the availability of this kit.
Figure 5. FAST-PETase part, optimized for E. coli
Figure 6. FAST-PETase + Linker + MHETase part, optimized for E. coli
Transformation of the DH5A competent cells with assembled plasmid by heat shock
In this step, we decided to use the transformation by heat shock method due to its ease of execution, which is the most used method for transformation in E. coli.
Colony PCR
In this step, our goal was to confirm the plasmid-transformed cells. Our primers were made with precision in mind to achieve accurate results, and the PCR had to be adapted to a better method for high GC content sequences.
Plasmid Miniprep
Our plasmid mini preps were planned to be made using the NEB Miniprep Kits and the Alkaline lysis method since they are simple, easy to execute, and effective.
BL21 competent cells transformation by heat shock method
For the expression of the plasmid we designed for E. coli, we had to transform the BL21 strain first since the backbone we chose to use was the most suitable for expression in this strain.
Expression induction with IPTG
The expression of the assembled enzymes in this test is IPTG induction dependent, so to express our FAST-PETase constructs, we had to add IPTG to the LB liquid cultures so that the transformed colonies could be incubated and later express our hydrolases.
Degradation
PCL degradation plate test
The last step for the E. coli part is the PCL degradation plate test, and it is the test we believe validates our construct in the sense of microplastic degradation, so in this stage, we had the orientation of our advisor, who shared articles so that we could understand it better.
Degradation test with polycaprolactone solid medium (PCL-LB)
For this test, we planned to do a PCL dispersion with acetone and incorporate this solution in the LB agar plate. When it's solid, we take an isolated colony from the expressing BL21 transformed plates and streak it in the degradation plate.
Results evaluation
Once the incubation period is over, the results to be observed are the formation of degradation halos around the grown colonies, so the confirmation of the expression can be seen with the naked eye.
This validation leads us to the next part of the planned experiments, the Chlamydomonas reinhardtii tests.
Part 2: Development stages for C. reinhardtii
| Stage | Experiments |
|---|---|
| 1. Preparation | Culture medium preparation (solid and liquid) |
| 1. Preparation | Reactivation and chemical competence evaluation tests of DH5A cells |
| 1. Preparation | Stock culture of the C. reinhardtii strains: CC1690, CC1691 e CC400 |
| 1. Preparation | CC-400 culture for experiments |
| 1. Preparation | pJP32PHL7 cloning with DH5A cells |
| 2. PHL7 expression | C. reinhardtii CC-400 with pJP32PHL7 transformation by glass beads method |
| 2. PHL7 expression | Colony PCR of pJP32PHL7 |
| 2. PHL7 expression | Degradation test with polycaprolactone solid medium (PCL-TAP) |
| 3. FAST-PETase Constructs Expression | pJP32PHL7 linearization by PCR |
| 3. FAST-PETase Constructs Expression | Assembly of the FAST-PETase and FAST-PETase_Linker_MHETase designed constructs with pJP32 linearized vector |
| 3. FAST-PETase Constructs Expression | Transformation of the DH5A competent cells with assembled plasmid by heat shock |
| 3. FAST-PETase Constructs Expression | Colony PCR |
| 3. FAST-PETase Constructs Expression | Plasmid Miniprep |
| 3. FAST-PETase Constructs Expression | C. reinhardtii transformation by glass beads method |
| 4. Degradation | Degradation test with polycaprolactone solid medium (PCL-TAP) |
| 4. Degradation | Results evaluation |
| 5. Toxicity | Culture of CC-400 strain in sewage |
| 5. Toxicity | Growth evaluation |
Table 2. Experiment Planning for Chlamydomonas reinhardtii
Preparation
Reactivation and chemical competence evaluation tests of DH5A cells
During this step, the goal is to use the reactivated cells from the previous step to clone the C. reinhardtii expression plasmid in the next steps.
Stock culture of the C. reinhardtii strains: CC1690, CC1691 e CC400
We made new striated plates and liquid cultivation of the three strains of C. reinhardtii.
CC-400 culture for experiments
The strain we chose to use for the tests was the CC-400 since it is a strain that doesn’t have a cell wall, and it facilitates the method of transformation that we chose.
PHL7 Expression
Since it was our first time working with Chlamydomonas reinhardtii and our constructs would arrive later, we decided to do the first tests with the pJP32PHL7 donated plasmid. The idea was to have a positive control since this plasmid contains the PHL7 enzyme, a widely known plastic-degrading hydrolase.
pJP32PHL7 cloning with DH5A cells
We chose the DH5a cells to execute the cloning tests with the pJP32PHL7 plasmid.
Figure 7. pJP32PHL7 donated plasmid
C. reinhardtii CC-400 with pJP32PHL7 transformation by glass beads method
With the Colony PCR and plasmid miniprep of the cloned DH5a, we would proceed to the transformation of C. reinhardtii with the glass bead method.
Degradation test with polycaprolactone solid medium (PCL-TAP)
After all the steps that confirmed the transformation's success, we planned to do a PCL dispersion with acetone and incorporate this solution in the TAP agar plate. When it's solid, we take an isolated colony from the CC-400 transformed plates and streak it in the degradation plate.
FAST-PETase Constructs Expression
The expression of FAST-PETase constructs in Chlamydomonas reinhardtii were planned to be tested in this step.
pJP32PHL7 linearization by PCR
To develop all the designs, we used Benchling to create primers and overlaps that could complement the C. reinhardtii expression vector sequence.
Figure 8. C. reinhardtii pJP32PHL7 plasmid linearization primers
Assembly of the FAST-PETase designed constructs with pJP32 linearized vector
In the same sense as the previous step, we wanted to use the Gibson Assembly Method and, alternatively, the Hi-Fi assembly method to assemble our constructs.
Figure 9. C. reinhardtii pJPFASTPETase and pJPFASTMHETase, respectively.
Transformation of the DH5A competent cells with assembled plasmid by heat shock
The transformation of the DH5A competent cells was planned to be performed by heat shock method to confirm the assembly and prepare the DNA to be transformed into the microalgae cells.
C. reinhardtii transformation by glass beads method
After performing Colony PCR and plasmid miniprep on the transformed cells, we would proceed to transform C. reinhardtii using the glass bead method.
Degradation
Once we confirm the success of the transformation, our next step would be to create a solution of PCL dispersion with acetone and mix it with the TAP agar plate. After the solution solidifies, we will transfer an isolated colony from the CC-400 transformed plates to the degradation plate. Once the incubation period is complete, we will observe the formation of degradation halos around the grown colonies. This will confirm the expression, which can be observed with the naked eye.
Toxicity
In the final stage of our planned tests with C. reinhardtii, we desired to conduct a test that would contribute to our implementation. In this sense, we planned to do a culture of the C. reinhardtii CC-400 strain in wastewater, with different concentrations of TAP medium, so we could be able to compare the growth of the microalgae.
Culture of CC-400 strain in sewage
The culture parameters for this test are detailed in the table below
| Medium | Wastewater | Medium |
| Tris-Acetate-Phosphate (TAP) | 10 mL | 90 mL |
| Tris-Acetate-Phosphate (TAP) | 25 mL | 75 mL |
| Tris-Acetate-Phosphate (TAP) | 50 mL | 50 mL |
| Tris-Acetate-Phosphate (TAP) | 75 mL | 25 mL |
| Tris-minimal | 10 mL | 90 mL |
| Tris-minimal | 25 mL | 75 mL |
| Tris-minimal | 50 mL | 50 mL |
| Tris-minimal | 75 mL | 25 mL |
| Tap water | 10 mL | 90 mL |
| Tap water | 25 mL | 75 mL |
| Tap water | 50 mL | 50 mL |
| Tap water | 75 mL | 25 mL |
| Waste water | 100 mL | 0 mL |
Table 3. C. reinhardtii Growth Test in Wastewater
Growth Evaluation
To evaluate the growth of the C. reinhardtii cells in these conditions, we would take a sample from the culture and add iodine solution to the sample, place 10 µL of it at each end of the Neubauer chamber, and count the cells using an optical microscope (40x magnification).
Stockholm-VIT Collaboration
We also were happy to collaborate with the Stockholm and VIT iGEM teams in the experiment they planned, a large-scale collaboration involving IGEM teams working on plastics and water pollution.
The plan was to create a map representing the microplastic levels in tap water in different regions of the world to generate more awareness about microplastics.
Check it out!
To reinforce the effort of working on this project, we invite you to access more information about our protocols on the Protocolos page.
For a detailed breakdown of our project's results and the implications of our findings, we invite you to dive into the data and insights that have shaped our project and discover our work's impact on synthetic biology. Visit our results page.
References
- Almeida, E. L., Carrillo Rincón, A. F., Jackson, S. A., & Dobson, A. D. (2019). In silico Screening and Heterologous Expression of a Polyethylene Terephthalate Hydrolase (PETase)-Like Enzyme (SM14est) With Polycaprolactone (PCL)-Degrading Activity, From the Marine Sponge-Derived Strain Streptomyces sp. SM14. Frontiers in Microbiology, 10, 476617. https://doi.org/10.3389/fmicb.2019.02187
- Economou, C., Wannathong, T., Szaub, J., Purton, S. (2014). A Simple, Low-Cost Method for Chloroplast Transformation of the Green Alga Chlamydomonas reinhardtii. In: Maliga, P. (eds) Chloroplast Biotechnology. Methods in Molecular Biology, vol 1132. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-995-6_27