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).

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.

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.

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.

Figure 2. Stock solutions for TAP medium

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.

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.

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.

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

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

Figure 8. NanoDrop Reading of the pJP32PHL7 MiniPrep

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.

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

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.

Figure 12. Wastewater + TAP medium cultures

Figure 13. Wastewater + Tris-minimal medium cultures

Figure 14. Wastewater + Tap water medium cultures

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.