iGEM 2023 — Team unila-latam

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*# Nootebook*
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[Week 1 (07/23/2023 - 07/29/2023)](#Week1)
[Week 2 (07/30/2023 - 08/05/2023)](#Week2)
[Week 3 (08/06/2023 - 08/12/2023)](#Week3)
[Week 4 (08/13/2023 - 08/19/2023)](#Week4)
[Week 5 (08/20/2023 - 08/26/2023)](#Week5)
[Week 6 (08/27/2023 - 09/02/2023)](#Week6)
[Week 7 (09/03/2023 - 09/09/2023)](#Week7)
[Week 8 (09/10/2023 - 09/16/2023)](#Week8)
[Week 9 (09/17/2023 - 09/23/2023)](#Week9)
[Week 10 (09/24/2023 - 09/30/2023)](#Week10)
[Week 11 (10/01/2023 - 10/06/2023)](#Week11)

## Week 1 (07/23/2023 - 07/29/2023)

Objective of the week: Reactivating competent cells with the RFP plasmid...

* **07/28**

  Testing the quimiocompetence INOUE method with DH5a cells

  Reactivation of DH10B cells that had the pSB1K3 plasmid

  Responsible: Jamily Brito Dorado and Bernie Reyes

## Week 2 (07/30/2023 - 08/05/2023)

Objective of the week: Testing our available competent cells for future clonings...

* **08/01**

  Inoculation of reactivated DH10B\_pSB1K3-RFP cells

  Cultivation of inoculated DH10B\_pSB1K3-RFP cells

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Cristian Rojas, and Bernie Reyes
* **08/02**

  Cultivation of INOUE\_DH5a cells

  Centrifugation of eppendofs with aliquots from the culture of DH10B\_pSB1K3-RFP cells...

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Cristian Rojas, and Bernie Reyes

## Week 3 (08/06/2023 - 08/12/2023)

Objective of the week: Design primers, continue the transformation process while monitoring...

* **08/06**

  Designing primers for colony PCR

  Responsible: Jamily Brito Dorado, Lucas Ovelar
* **08/07**

  Verify the cell growth in petri dishes

  Purification of the minipreps made in 08/03

  Responsible: Jamily Brito Dorado, Matheus Araújo, and Bernie Reyes
* **08/08**

  Continuation of the purification process

  Nanodrop readings

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **08/09**

  NanoDrop readings of the minipreps

  New inoculum culture for new miniprep assay

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **08/10**

  Miniprep using homemade kit

  Miniprep using NEB kit

  NanoDrop readings

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Matheus Araújo, and Bernie Reyes
* **08/11**

  Preparing transformation plates

  Preparing minipreps for transformation

  NanoDrop readings

  Responsible: Jamily Brito Dorado and Bernie Reyes

## Week 4 (08/13/2023 - 08/19/2023)

Objective of the week: Evaluation of the transformations based on the transformation of chemically competent cells...

* **08/14**

  Aliquoting the plasmid pSB1K3

  Resuspension of the plasmid pJP32PHL7

  NanoDrop readings of the pJP32PHL7 resuspension

  Chemically competent cells transformation with pSB1K3 using the heat shock method

  Chemically competent cells transformation with pJP32PHL7 using the heat shock method

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **08/15**

  Evaluating transformation plates

  Preparing plates for culturing

  Preparing tubes for inoculum

  Antibiotic quotation (Zeocin/Bleomicin)

  Responsible: Jamily Brito Dorado and Bernie Reyes
* **08/16**

  Inoculum of the transformed cells (pJP32PHL7)

  Responsible: Jamily Brito Dorado
* **08/17**

  pJP32PHL7 miniprep

  Aliquoting and storing the miniprep

  Responsible: Jamily Brito Dorado, Thaynara Castro, and Bernie Reyes
* **08/18**

  We received the strains CC-400, CC-1690, and CC-1691, kindly donated by professor Lívia Seno...

  Identifying restriction regions of the pJP32PHL7 plasmid

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

## Week 5 (08/20/2023 - 08/26/2023)

Objective of the week: Preparing the TAP media for culturing our donated Chlamydomonas reinhardtii strains and continuing to verify the pJP32PHL7 resuspension.

* **08/21**

  NanoDrop readings (pJP32PHL7)

  Enzyme digestion protocol (NEB) familiarization

  Organizing and obtaining the ingredients for TAP medium preparation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Ghadir Wissam Rizk, Bernie Reyes, and Cristian Rojas
* **08/22**

  NanoDrop readings (pJP32PHL7)

  Enzyme digestion protocol execution

  Agarose gel electrophoresis with the digestion result

  IDT sequences order

  Electrophoresis gel results analysis

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Ghadir Wissam Rizk, Bernie Reyes, and Cristian Rojas
* **08/23**

  TAP medium ingredients were obtained and separated

  Agarose gel electrophoresis with the digestion result

  Responsible: Cristian Rojas
* **08/24**

  Preparation of TAP medium stock solutions

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Ivan Mateo Espitia, Thaynara Castro, Bernie Reyes, and Cristian Rojas
* **08/25**

  Electrophoresis gel results analysis

  TAP medium stock solutions pH adjustments

  New streaking plates with pHP32PHL7 E. coli colonies

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Ivan Mateo Espitia, and Cristian Rojas
* **08/26**

  We kindly received a donation of the antibiotic zeocin from Wesley Luzetti Fotoran, a colleague USP student

  Responsible: Lucas Ovelar

## Week 6 (08/27/2023 - 09/02/2023)

Objective of the week: Preparing the TAP media for culturing our donated Chlamydomonas reinhardtii strains and continuing to verify the pJP32PHL7 resuspension.

* **08/28**

  pJP32PHL7 streak plates evaluation

  New solid medium plates preparation

  1L TAP medium preparation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **08/29**

  C. reinhardtii CC-400, CC-1690 and CC1691 inoculum with TAP medium

  DH10B cells with pJP32PHL7 inoculum

  LB liquid medium preparation

  LB solid medium preparation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **08/30**

  C. reinhardtii strains inoculum growth verification

  Responsible: Jamily Brito Dorado
* **08/31**

  Alkaline lysis miniprep execution

  Homemade miniprep execution

  Miniprep solutions preparation

  NanoDrop readings

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **09/01**

  C. reinhardtii strains inoculum growth verification

  Responsible: Jamily Brito Dorado

## Week 7 (09/03/2023 - 09/09/2023)

Objective of the week: Purify our minipreps to confirm that we obtained the pJP32PHL7 plasmid, isolate the Chlamydomonas reinhardtii cells from contaminated culture and results analysis.

* **09/04**

  Ethanol precipitation of RNA/DNA protocol execution

  C. reinhardtii strains inoculum growth verification: contaminated

  New C. reinhardtii inoculums with TAP medium

  Miniprep NanoDrop readings

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **09/05**

  Agarose gel electrophoresis preparation

  Contaminated crops were placed next to a lamp so that C. reinhardtii cells migrated towards the light and isolated

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/06**

  Agarose gel electrophoresis

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **09/07**

  Agarose gel results analysis

  C. reinhardtii culture results analysis

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

## Week 8 (09/10/2023 - 09/16/2023)

Objective of the week: Culturing our donated Chlamydomonas reinhardtii strains, evaluating the dye reagents we had available to verify their functioning, as well as verifying the success of the minipreps made throughout the weeks using samples already known as a comparison parameter.

* **09/11**

  pJP32PHL7 transformation assay

  Agarose gel electrophoresis to verify the samples and dyes

  C. reinhardtii culture verification

  C. reinhardtii cell counting

  Degradation test plates reagents organization and quotation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/12**

  pJP32PHL7 cloning assay

  Miniprep protocol evaluation

  Agarose gel dyes results evaluation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes
* **09/13**

  Agarose gel electrophoresis with different concentrations of dye and successful DNA samples

  Agarose gel results analysis

  Microplastic dispersion research

  Responsible: Jamily Brito Dorado and Cristian Rojas
* **09/14**

  pJP32PHL7 transformation using the heat shock method with donated competent cells from PTI and our available DH5a cells

  Agarose gel electrophoresis with different concentrations of Diamond Dye and TAE buffer

  Agarose gel analysis by transluminator results

  TAP medium preparation

  Responsible: Jamily Brito Dorado, Thaynara Castro, Lucas Ovelar, and Bernie Reyes
* **09/15**

  Agarose gel electrophoresis with different 1:100 concentration of Diamond Dye and TBE buffer

  Agarose gel analysis by transluminator results

  Antifungal plate with TAP medium preparation

  Structuring the Chlamy Manual

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

## Week 9 (09/17/2023 - 09/23/2023)

Objective of the week: Culturing our donated Chlamydomonas reinhardtii strains, continuing to verify the pJP32PHL7 resuspension, and executing a collaboration protocol with other iGEM teams.

* **09/18**

  Transformation plates with pJP32PHL7 and PTI competent cells were evaluated and with good results

  Inoculum preparation for miniprep

  C. reinhardtii culture verification - contaminated

  New streak plates on TAP solid medium with all the C. reinhardtii strains available

  Streaking C. reinhardtii in plates prepared previously with antifungal and TAP medium

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/19**

  Miniprep protocol execution

  Check plates with pJP32 PTI transformed cells

  LB medium plates preparation for G0 plasmid transformation

  Polyurethane dispersion quotation

  iGEM Stockholm x iGEM VIT Vellore Collaboration protocol execution

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/20**

  NanoDrop readings

  Results evaluation

  Next steps analysis

  Responsible: Lucas Ovelar and Bernie Reyes
* **09/21**

  TE buffer preparation

  EDTA solution preparation

  Agarose gel electrophoresis

  Chemically competent cells quotation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/22**

  Reviewing all transformation assays and analyzing results

  Lab next steps organization and planning

  Agarose gel electrophoresis

  Agarose gel electrophoresis results evaluation

  Responsible: Jamily Brito Dorado, Lucas Ovelar, and Bernie Reyes

## Week 10 (09/24/2023 - 09/30/2023)

Objective of the week: Reactivation and linearization of G0 and pJP32PHL7 plasmids, resuspending IDT primers, optimizing lab work, and planning next steps based on our meeting with João Molino (Advisor).

* **09/25**

  TE 10X pH verification

  Setting a meeting with our advisor João Molino

  Lambda digestion with PstI

  IDT primers resuspensions

  G0 reactivation

  Responsible: Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/26**

  Organization of the week

  Polystyrene dispersion quotation

  Responsible: Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/27**

  pJP32PHL7 linearization PCR

  Agarose gel electrophoresis

  C. reinhardtii new plates re-streak

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/28**

  Agarose gel plasmid extraction

  Streaking CC-400 strain to carbanzandin plates with different concentrations

  Culturing CC-400 strain into new liquid TAP medium

  Inoue method testing assay

  Responsible: Jamily Brito Dorado, Lucas Ovelar, Bernie Reyes, and Cristian Rojas
* **09/29**

  Inoue method execution

  Advisor meeting

  Policaprolactone purchase

  Preparation of materials for culturing CC-400 strain in sewage with different concentrations and mediums

  TAP tris minimal preparation

  Responsible: Jamily Brito Dorado
* **09/30**

  Policaprolactone purchase

  Preparation of materials for culturing CC-400 strain in sewage with different concentrations and mediums

  TAP tris minimal preparation

  Responsible: Jamily Brito Dorado

## Week 11 (10/01/2023 - 10/06/2023)

Objective of the week: Culturing the strain CC-400 in different concentrations of sewage and medium, verifying growth in the carbandanzin plates with CC-400 strain, and linearizing the pJP32PHL7 and G0 plasmids.

* **10/02**

  Autoclaving materials for sewage culture

  pJP32PHL7 linearization PCR with different polymerases

  Agarose gel electrophoresis

  CC-400 growth verification in carbandanzin plates

  CC-400 inoculum in cultures with different concentrations of sewage

  Responsible: Jamily Brito Dorado, Cristian Rojas, and Bernie Reyes
* **10/03**

  Agarose gel electrophoresis with PCR products and different samples for comparison

  Agarose gel results analysis

  Microplastic dispersion research

  Responsible: Jamily Brito Dorado and Cristian Rojas
* **10/04**

  G0 linearization PCR

  LB with kanamycin plates preparation

  Agarose gel electrophoresis preparation

  Responsible: Jamily Brito Dorado
* **10/05**

  Agarose gel electrophoresis with linearization results

  Results analysis - negative

  G0 linearization PCR

  Agarose gel electrophoresis with linearization results

  Responsible: Jamily Brito Dorado

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*# Engineering*
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[Introduction](#Introduction)
[Genetic Circuit](#Genetic)
[Implementation](#Implementation)
[Enzyme choice](#Enzyme)
[Model](#Model)
[Conclusion](#Conclusion)
[Future Perspective](#Future)

# Introduction

The development of Mhetyguá’s project was meticulously grounded in solid engineering practices to optimize both the progression of the project and the excellence of the end product. Throughout the project, we employed several iterative engineering cycles, encompassing design, build, test, and learn phases, to establish a dependable system. Leveraging these principles, we effectively constructed our project for microplastic degradation. A more detailed breakdown of these guiding principles and steps can be found on the subsequent page, with a special emphasis on its application in Chlamydomonas Reinhardtii. We also talked to stakeholders and researchers to gain knowledge we otherwise would not have obtained. These warranted repeated changes to our design and tweaks to aspects of our project.

## Genetic Circuit

We recommend reading the [design](https://2023.igem.wiki/unila-latam/design) page first to understand the context of our genetic circuit design and how the DBTL cycle was applied.

### Cycle 1

* **Design:** The initial design of our circuit was intricate and expansive, employing the Golden Gate assembly methodology. This design was informed by the research conducted by prior iGEM teams and pertinent scientific literature in the domain.
* **Build:** Utilizing Benchling, we engineered the primary circuit, incorporating essential modifications to ensure effective expression in both Chlamydomonas reinhardtii and Escherichia coli.
* **Test:** The assembly was simulated using Benchling to evaluate its feasibility.
* **Learn:** Post in-silico evaluations indicated that the assembly process would be protracted, given the multitude of assemblies planned and the anticipated delivery timeline of our syntheses. Consultations with our advisors, João Molino, Samuel Chagas, Matheus Araujo, Giulio Bratz, and our Principal investigator, Cristian Rojas, led to the consensus that a more streamlined circuit, retaining the capability to express our target enzyme, would be more pragmatic.

### Cycle 2

* **Design:** The conceptualization for our secondary circuit was influenced by prior research undertaken by our advisor, João Molino. He advocated for a more simplified circuit design. Concurrently, our lead researcher, Cristian Rojas, recommended the adoption of the Gibson Assembly methodology for our assemblies, given the pre-existing laboratory resources compatible with this technique, eliminating the need for additional acquisitions of reagents.
* **Build:** The circuits were meticulously designed in Benchling, tailored for both Chlamydomonas reinhardtii and Escherichia coli. Presently, we are in the advanced stages of assembling our plasmids embedded with the circuit.
* **Test:** To be conducted in the imminent future.
* **Learn:** A salient insight we have gleaned thus far is the occasional necessity for minimalistic approaches, especially when constrained by time. Such an approach ensures the optimization of our scientific endeavors.

## Implementation

### Cycle 1

* **Design:** At the beginning of our research, we identified the possibilities where the project could be implemented, considering our chassis, releasing it into a body of water like a river was not an option, due to biosecurity concerns. So we decided that our implementation would be inside a photobioreactor.
* **Build:** Since we don't have a photobioreactor in our laboratory, we would have to buy or build one. The option to buy was not viable due to economic constraints. So we decided to build a photobioreactor, for which we contacted Victor Marchesan, who developed the Open-Source Modular Bioreactor, hardware from the 2022 iGEM UFMG\_UFV team.
* **Test:** Following discussions with individuals experienced in photobioreactor design, we formulated a theoretical prototype and made a budget for purchasing the parts.
* **Learn:** We realized that building a photobioreactor was not a priority, as we would have to cover the costs of the entire competition, even though the budget was not large, we needed to save the money. Furthermore, the need to advance our research to innovate in the development of a homemade PBR became evident, given that certain iGEM teams had already successfully created such hardware. Given these constraints, both resources and time were not in our favor.

### Cycle 2

* **Design:** After seeing the infeasibility of building our photobioreactor, we explored alternative methods for facilitating enzymatic degradation. As a result, we considered the possibility of enzyme immobilization.
* **Build:** We conducted research on enzyme immobilization techniques and determined that applying enzymes within alginate beads, a material frequently employed for this purpose, was the most suitable approach. The ultimate implementation of this method would be within water treatment plants.
* **Test:** Upon reaching out to the local sanitation institution, we were informed that employing biological techniques for water treatment might not be the most suitable approach, as they primarily rely on physical-chemical methods. Consequently, they recommended the use of immobilized enzymes for sewage treatment, where biological methods were already in practice.
* **Learn:** Following discussions with stakeholders, we also conducted a comprehensive economic analysis and determined that a more cost-effective approach would be to directly apply the microalgae to the sewage treatment plant. This would allow the microalgae to secrete the enzymes naturally, eliminating the need for enzyme isolation, which tends to increase process costs.

### Cycle 3

* **Design:** Finally, we directed our research towards a more specific and contextual area. It became evident that implementing our solution in wastewater treatment plants was the most practical choice. We identified stabilization ponds as the optimal location for our project implementation, given that these ponds naturally support the growth of microorganisms like microalgae.
* **Build:** We focused on understanding the operational dynamics of stabilization ponds, in addition to seeking data on the socio-environmental context and the utilization of this sewage treatment approach in Brazil.
* **Test:** To conduct a proof of concept, we cultivated our microalgae in TAP culture medium with samples of wastewater at different concentrations (10%, 25%, 50%, 75%, and 100%).
* **Learn:** We are currently in the phase of analyzing the growth rate in these conditions to later compare it with the widely used TAP culture medium and define new actions for the project.

## Enzyme choice

### Cycle 1

* **Design:** In the early stages of our research, we embarked on a quest to identify the most suitable enzyme for PET degradation. We encountered several options, including PETase, LCC, and PHL7. To make an informed choice, we conducted thorough comparisons among them (check our Design page [here](https://2023.igem.wiki/unila-latam/design)), meticulously evaluating the respective advantages and disadvantages associated with each enzyme.
* **Build:** We finally opted for PHL7 due to its remarkable stability and superior PET degradation rate, especially at elevated temperatures like 70°C. This decision was aligned with our initial implementation concept, which involved a bioreactor setup. Consequently, we proceeded to construct our circuit incorporating PHL7.
* **Test:** Since we didn't have any plasmid that expressed PHL7, we were looking for a suitable one. Fortunately, our advisor, João Molino, generously donated one to our project. This proved to be immensely beneficial, saving us from the 45-day waiting period typically associated with obtaining a synthesis from IDT.
* **Learn:** Throughout the progression of our project, we underwent several iterations in our implementation strategy. These adjustments significantly influenced our selection of the enzyme of interest. Notably, as we shifted our focus to the utilization of a stabilization pond, it became evident that PHL7 would no longer operate at its peak catalytic efficiency since the temperature within the stabilization pond is lower than the enzyme's optimal 70°C operating range.

### Cycle 2

* **Design:** Considering the final implementation idea (check our implementation [here](https://2023.igem.wiki/unila-latam/implementation)), we explored alternative enzymes and came across FAST-PETase, an enzyme notable for its commendable catalytic rate in PET degradation, particularly well-suited for the temperature typically found in stabilization ponds.
* **Build:** Considering the new enzyme, FAST-PETase, we proceeded to redesign our circuit, optimizing it for Chlamydomonas reinhardtii and Escherichia Coli, then subsequently we built our plasmid based on the previous construction. Essentially, our design only entailed replacing PHL7 with FAST-PETase.
* **Test:** We are in the phase of building our plasmid, first linearizing our plasmids using PCR and then assembling them with Gibson Assembly's FAST-PETase.
* **Learn:** After building our plasmid we will move on to the analysis phase of the catalytic activity and the expression rate of this enzyme to then compare it with PHL7 and define new actions for the project.

## Model

### Cycle 1

* **Design:** Our first modeling idea came from the concept of ecological modeling, which incorporates a variety of ecological factors such as species interactions, energy flow, nutrient cycles, population dynamics, and climate change.
* **Build:** The aim was to determine the ultimate biomass of our microalgae by constructing a model that delineates interactions between species.
* **Test:** While the concept of ecological modeling intrigued us, we found it unsuitable for the initial phase of the project. This decision was influenced by biosafety and implementation concerns, as well as the fact that it was an entirely new area for our team members, this posed potential challenges given our limited time frame. Furthermore, we lacked a network of individuals experienced in this specific modeling field who could provide guidance and support.
* **Learn:** We gained familiarity with fundamental ecological modeling concepts that could be integrated into other modeling methods, which we had previously had contact with.

### Cycle 2

* **Design:** With the implementation of the project aimed at stabilization ponds, we had the idea of modeling something that involved this topic, especially because other iGEM teams had already worked with PET degrading enzymes in microalgae, so we would like to design our unique model. So, we decided to kinetically model the simultaneous growth of our microalgae and the PET degradation in sewage, while also structurally modeling an enzyme-linker complex.
* **Build:** Overall, a lot of literature research was necessary, as well as inspiration from models similar to the context of our project. To develop the kinetic and structural analyses, we used computational resources, in addition to previously described data and equations.
* **Test:** As we carried out our simulations, some adaptations were necessary so that we could reach more satisfactory and coherent results. For example, the first analyzes of the graphs completely changed the integration structure of our model, moving from differential equations to the Gompertz model. If you want to understand the modeling process and more adaptations done, check our Model page [here](https://2023.igem.wiki/unila-latam/model).
* **Learn:** In the example mentioned above, we understood that our modifications to the base model we used directly influenced the final analysis, which made us insert a growth model based on the sigmoidal microalgae growth curve to generate truly meaningful comparisons.

# Conclusion

The Mhetygua project showcases a systematic and iterative approach to addressing the challenge of microplastic degradation. By employing sound engineering practices, the team navigated through various phases of design, build, test, and learn, ensuring that each step was grounded in research, expert advice, and practical considerations. The project's adaptability was evident in the shifts in implementation strategies, from photobioreactors to wastewater treatment plants, and in the enzyme choices, transitioning from PHL7 to FAST-PETase based on efficiency and environmental conditions.

# Future Perspective

Moving forward, the Mhetygua project holds significant promise in the realm of environmental biotechnology. As the team continues to analyze the catalytic activity and expression rate of FAST-PETase, there is potential for further optimization and scalability. Collaborations with wastewater treatment facilities could pave the way for real-world applications, transforming the way we address microplastic pollution. Additionally, the project serves as a blueprint for future iGEM teams and researchers, emphasizing the importance of adaptability, stakeholder engagement, and iterative design in scientific endeavors. As the global community becomes increasingly aware of environmental challenges, projects like Mhetygua will be at the forefront of sustainable solutions.

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*# Experiments*
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[Introduction](#Introduction)
[Learning about the microalgae](#Learning)
[Obtaining the strains and reagents](#Obtaining)
[Experiment Planning](#Experiment)
[Work Cells](#Work)
[Glass Beads Transformation](#Glass)
[Plastic Degradation](#Plastic)
[Part 1: Development stages for E. coli](#Part1)
[Preparation and Cloning](#Preparation)
[Reactivation and chemical](#Reactivation)
[E. coli expression plasmid cloning with DH5A cells](#Coli)
[E. coli expression plasmid linearization by PCR](#Plasmid)
[Expression](#Expression)
[FAST-PETase and FAST-PETase\_Linker\_MHETase](#Fast)
[Assembly of the FAST-PETase](#Assembly)
[Transformation of the DH5A](#Transformation)
[Colony PCR](#Colony)
[Plasmid Miniprep](#Miniprep)
[BL21 competent cells transformation](#BL21)
[Expression induction with IPTG](#Induction)
[Degradation](#Degradation)
[PCL degradation plate test](#PCL)
[Degradation test with polycaprolactone solid medium (PCL-LB)](#Polycaprolactone)
[Results evaluation](#Results)
[Part 2: Development stages for C. reinhardtii](#Part2)
[Preparation](#Preparation)
[Reactivation and chemical competence evaluation tests of DH5A cells](#Chemical)
[Stock culture of the C. reinhardtii strains: CC1690, CC1691 e CC400](#Stock)
[CC-400 culture for experiments](#CC-400)
[PHL7 Expression](#PHL7)
[pJP32PHL7 cloning with DH5A cells](#pJP32PHL7)
[C. reinhardtii CC-400 with pJP32PHL7 transformation by glass beads method](#reinhardtii)
[Degradation test with polycaprolactone solid medium (PCL-TAP)](#solid)
[FAST-PETase Constructs Expression](#Constructs)
[pJP32PHL7 linearization by PCR](#linearization)
[Assembly of the FAST-PETase designed constructs](#constructs)
[Transformation of the DH5A](#competent)
[C. reinhardtii transformation by glass beads method](#method)
[Degradation](#Degradation2)
[Toxicity](#Toxicity)
[The culture parameters](#parameters)
[Growth Evaluation](#Growth)
[Stockholm-VIT Collaboration](#Stockholm)
[Check it out!](#Check)
[References](#References)

## 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](https://2023.igem.wiki/unila-latam/contribution).

## 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.

![Imagen Centrada y Redimensionada](https://static.igem.wiki/teams/5023/wiki/experiments-page/image11.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image6.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image2.png)
![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image8.png)

**Figure 3. E. coli expression plasmid + FAST-PETase linearization primers**

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image7.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image1.png)

**Figure 5. FAST-PETase part, optimized for E. coli**

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image3.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image4.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image5.png)
![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image10.png)

**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.

![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image9.png)
![](https://static.igem.wiki/teams/5023/wiki/experiments-page/image12.png)

**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>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

[Introduction](#Introduction)
[Preparation (E. coli)](#Preparation)
[Preparation (C. reinhardtii)](#Preparation2)
[FAST-PETase Constructs Expression](#Fast)
[The Enigma of the Missing Plasmid pJP32PHL7](#Enigma)
[Glass Beads Transformation](#Glass)
[Conclusion](#Conclusion)

# Introduction

Our team recognizes the significance of illustrating the full scope and potential of our project in the scientific community. Ideally, a thorough proof of concept should not only demonstrate its practicality but also its innovative approach in the field of biotechnology.

Nevertheless, due to the constraints of time, the cancellation of the IDT gBlock orders (FAST-PETase\_Linker\_MHETase gBlock) for E. coli, and the late arrival of the ordered synthesis for C. reinhardtii and E. coli, we couldn’t proceed with our planned sequence of experiments and had to strategically concentrate our research and experimentation on the Chlamydomonas reinhardtii part (Part 2).

![](https://static.igem.wiki/teams/5023/wiki/results-page/images1.png)

**Table 1. Experiments Execution**

Our experiments were developed in 4 stages that mainly involved: preparations, cloning pJP32PHL7 plasmid, PHL7 expression in C. reinhardtii, linearization of the pJP32PHL7 plasmid, FAST-PETase and FAST-PETase\_Linker\_MHETase expression in C. reinhardtii and toxicity test.

## Preparation (E. coli)

### Reactivation and chemical competence evaluation tests of DH5A cell.

We tested various chemically competent cells to rule out the possibility that they are neutral competent cells. For this, we conducted several experiments.

![](https://static.igem.wiki/teams/5023/wiki/results-page/images2.png)

**Table 2. Chemically Competent Cells Evaluation**

In this table we summarize all the competent cell test results we had, the Commercial cells were the bought cells we had available in the lab, and the PTI cells were donated by our PI’s colleagues who work at the Itaipú Technological Park labs, and the EGE cells were the ones made with the INOUE method.

These results were compiled in several weeks of evaluation of each strain, to achieve a positive control as a comparison parameter for future transformations with our designed constructs alongside the other experiments planned.

### E. coli expression plasmid linearization by PCR

Upon arrival of the IDT synthesis, we attempted to linearize the E. coli expression plasmid to better understand and improve our constructs despite our limited time.

![Imagen Centrada y Redimensionada](https://static.igem.wiki/teams/5023/wiki/results-page/image13.jpg)

**Figure 1. Linearization agarose gel electrophoresis results.**

Our results did not align with our expectations. Unfortunately, we were unable to review our design in a timely manner, leaving open the possibility that the adapted primers may have deviated from the original design in terms of accuracy.

## Preparation (C. reinhardtii)

### Culture medium preparation (solid and liquid)

In our initial attempt at working with C. reinhardtii, we took care to begin with the basics, including preparing the appropriate culture medium for this microalgae. We thoroughly verified the purity of all essential TAP medium ingredients and then went on to ensure complete sterilization of both the instruments and medium. Dilutions were accurately made from concentrated solutions, and the pH level of the medium was then adjusted to a desirable value of approximately 7. Following autoclaving, the medium was appropriately stored, resulting in a top-quality medium that facilitated optimal growth of Chlamydomonas reinhardtii in our experiments.

![Imagen Centrada y Redimensionada](https://static.igem.wiki/teams/5023/wiki/results-page/image4.png)

**Figure 2. Stock solutions for TAP medium**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image3.png)

**Figure 3. pH control of TAP medium**

### Stock culture of the C. reinhardtii strains: CC1690, CC1691 e CC400

Our initial phase involved setting up the optimal conditions for culturing Chlamydomonas reinhardtii. We meticulously calibrated factors such as light intensity, nutrient concentration, and pH levels to ensure a conducive environment for growth. Over time, we observed that these microalgae exhibited a robust growth rate under specific conditions, which we documented for future reference. However, like any scientific endeavor, we faced challenges. Contamination was an occasional issue, and understanding the precise nutritional needs of Chlamydomonas reinhardtii required iterative testing. However, these hurdles provided valuable learning experiences, refining our methodologies and enhancing our understanding of microalgal biology.

![](https://static.igem.wiki/teams/5023/wiki/results-page/image2.jpg)

**Figure 4. Chlamydomonas reinhardtii strains culture**

### CC-400 culture for experiments

The chosen strain to perform all the experiments with was the CC-400, so we dedicated a new culture for these innoculums so that we could work with the ones that had better growth.

![](https://static.igem.wiki/teams/5023/wiki/results-page/image1.jpg)

**Figure 5. Cultures of CC-400 strain**

Our culturing of the strain CC-400 had satisfactory results for its purpose.

### pJP32PHL7 cloning with DH5A cells

Once our CC400 strains are ready, it's time to transform them, but first, we need the plasmid to carry out the transformation. For this, we recovered the pJP32PHL7 plasmid and then transformed it into an E. coli DH5A. Initially, we thought that the colonies that grew were the ones that integrated the plasmid with ampicillin resistance.

![](https://static.igem.wiki/teams/5023/wiki/results-page/image5.png)

**Figure 6. Plate with E. coli colonies apparently transformed.**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image14.png)

**Figure 7. Verification of growth of DH5a and DH10b in ampicillin**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image12.png)

**Figure 8. NanoDrop Reading of the pJP32PHL7 MiniPrep**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image11.jpg)

**Figure 9. Agarose gel electrophoresis results**

Based on the outcomes we obtained, we initially believed that we had accomplished the cloning of the pJP32PHL7 plasmid. Nevertheless, upon performing agarose gel electrophoresis and endeavoring to linearize the plasmid, we discovered that it was not present.

## FAST-PETase Constructs Expression

### pJP32PHL7 linearization by PCR

When the resuspension of the donated plasmid was ineffective in cloning tests, we immediately attempted to linearize the pJP32PHL7 plasmid upon the arrival of our IDT synthesized parts.

![](https://static.igem.wiki/teams/5023/wiki/results-page/image9.jpg)

**Figure 10. Agarose gel electrophoresis results using different forms of target plasmids and control samples**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image10.jpg)

**Figure 11. Agarose gel electrophoresis results after PCR linearization of the putative pJP32PHL7, our plasmid samples are in wells 2 and 3**

Despite conducting multiple experiments and employing different techniques to convert the circular pJP32PHL7 plasmid into a linear form, we were unable to obtain desirable outcomes during the agarose gel electrophoresis analysis.

### Culture of CC-400 strain in sewage

During the experiment, we noticed that the conventional Chlamydomonas culture did not exhibit the typical green color. However, we did observe a slight change in the coloring of the medium, which appeared to be visually 'cleaner'. In comparison to the erlenmeyers that contained wastewater samples, some of the bottles appeared to have a whiter medium, while others appeared to have a more transparent medium.

![](https://static.igem.wiki/teams/5023/wiki/results-page/image15.jpg)

**Figure 12. Wastewater + TAP medium cultures**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image8.jpg)

**Figure 13. Wastewater + Tris-minimal medium cultures**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image7.jpg)

**Figure 14. Wastewater + Tap water medium cultures**

![](https://static.igem.wiki/teams/5023/wiki/results-page/image6.jpg)

**Figure 15. Wastewater samples**

### Growth evaluation

The evaluation of the growth rates in these cultures is made by counting the cells of each sample in the Neubauer chamber. Lamentably we weren’t able to count these samples properly because of time limitations and a shutdown that happened at our university.

# The Enigma of the Missing Plasmid pJP32PHL7: A Discussion on Potential Scenarios

The absence of the plasmid pJP32PHL7 in our laboratory experiments has raised several questions and speculations. Given the critical role this plasmid plays in our research, its absence has had a profound impact on our experimental outcomes. As we reflect on the possible reasons for this missing component, a few scenarios emerge that might explain the mystery surrounding the elusive pJP32PHL7.

* **Degradation Prior to Arrival:**One plausible explanation is that the plasmid might have degraded before even reaching our laboratory. Plasmids, like other DNA molecules, are susceptible to degradation under certain conditions. Factors such as exposure to elevated temperatures, enzymatic activity, or even prolonged transit times can compromise the integrity of the plasmid. If the plasmid was shipped to our laboratory, it's conceivable that it might have encountered conditions unfavorable for its stability during transit.
* **Compromised Extraction Process:**The process of extracting plasmids is intricate and requires precision. Any deviation from the established protocol, be it in terms of reagent quality, equipment calibration, or even human error, can adversely affect the outcome. It's possible that during the extraction process, the plasmid suffered damage that rendered it undetectable or non-functional. DNA can be sheared or broken if subjected to excessive mechanical forces, and contaminants introduced during the extraction can also interfere with the recovery of the plasmid.

In conclusion, while the exact reason for the absence of the plasmid pJP32PHL7 remains speculative, the scenarios discussed above offer plausible explanations. The nature of scientific research is such that unexpected challenges often arise, and the case of the missing plasmid underscores the importance of meticulous attention to detail at every step of the experimental process. As we move forward, lessons learned from this experience will undoubtedly inform our future endeavors and protocols.

# Glass Beads Transformation

Unfortunately, we were unable to carry out the glass beads transformation procedure for Chlamydomonas reinhardtii, as we did not manage to obtain our pJP32PHL7 plasmid. This setback has been a learning experience for our team. While we had anticipated promising results based on the potential of the glass beads method to facilitate the uptake of foreign DNA into the Chlamydomonas reinhardtii cells, we were unable to validate these expectations due to the absence of the necessary plasmid.

Despite this challenge, our team remains optimistic about the potential of the glass beads method for genetic engineering within this microorganism in our lab. We believe that once we secure the required plasmid and resources, we can revisit this method and potentially establish it as a standard practice for Chlamydomonas reinhardtii transformations in the future.

# Conclusion

The journey of the UNILA-LatAm team in the realm of synthetic biology, particularly in the degradation of plastic using FAST-PETase, was marked by a series of challenges that impacted the progression and outcomes of our experiments. One of the most significant hurdles we faced was the prolonged search for the essential plasmid, pJP32PHL7.

The acquisition of this plasmid proved to be a daunting task. Despite our relentless efforts, reaching out to various universities and professionals, and even considering purchasing options, the quest for pJP32PHL7 consumed a considerable amount of our project timeline. While we were fortunate to receive guidance and contacts from our advisor João Molino, and eventually a donation of the strains we needed, the time and resources expended in this pursuit were substantial.

This extended search for the plasmid, coupled with the challenges of working with a novel organism, meant that a significant portion of our project timeline was dedicated to preliminary preparations rather than the core objective of testing plastic degradation with FAST-PETase. Furthermore, the construction of our subsequent genetic circuits was intrinsically linked to the availability and functionality of the pJP32PHL7 plasmid. Without this foundational component, advancing to the next stages of our project became untenable.

Additionally, venturing into uncharted territory by working with Chlamydomonas reinhardtii presented its own set of challenges. As the first team at our university to delve into research with this microalgae, we lacked the institutional knowledge and experience that often streamline experimental processes. The absence of prior work with Chlamydomonas reinhardtii meant that we were pioneering not only the methodologies but also establishing foundational knowledge about the organism within our academic community.

While our team was driven by a clear vision and unwavering dedication, the challenges associated with procuring the pJP32PHL7 plasmid and pioneering work with Chlamydomonas reinhardtii significantly impacted our ability to achieve the expected objectives. The intricacies of scientific research often present unforeseen challenges, and in our case, the combined complexities of working with a new organism and the dependency on a single plasmid underscored the intricacies of advancing in the field of synthetic biology.

This project wasn't just a milestone for our research group but also a beacon for the broader university community. As the first research group at our university to delve into the intersection of microalgae and genetic engineering, we've blazed a trail for subsequent research endeavors. We've equipped our laboratory with invaluable resources, including gBlocks tailored for Chlamydomonas reinhardtii, three distinct strains of C. reinhardtii, specialized culture media, and other essential tools for microalgae research. This initiative ensures that other students and researchers at our university have a solid foundation to build upon, fostering an environment of innovation and exploration in the realm of microalgae research.

The results have paved the way for further exploration into the vast potential of microalgae in synthetic biology applications. Our pioneering work with Chlamydomonas reinhardtii has not only yielded promising results but also established a robust framework for future research, ensuring that our university remains at the forefront of microalgal biotechnology.

Although our results did not come out completely as expected, we couldn’t have come this far without our advisors’ and PI’s help, their guidance and knowledge led us to learn and create a new perspective to continue this project as it was planned.

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