First and foremost, our journey began with the quest to understand what we were seeking. We aimed to tackle the issue of the emergence of super bacteria, a significant and increasingly perilous problem over time. This endeavor posed several fundamental questions: How can we develop superior antibiotics, and how can we enhance their production?

During our initial research, we unearthed a pivotal discovery - Streptomyces species were responsible for producing a staggering 80% of all antibiotics [1]. This revelation led us to contemplate our first essential step in addressing this significant challenge: the need to enhance this microorganism and transform it into the world's largest antibiotic factory. However, a substantial hurdle lay in our path - Streptomyces is a challenging bacterium to work with. It is poorly documented in the scientific literature, and its mechanisms remain enigmatic.

With this in mind, we concluded that our first stride towards addressing this concern and contributing to averting a potential catastrophe was to "simplify working with this chassis, collaborate with fellow scientists, and transform Streptomyces into the new E. Coli."

With these thoughts in mind, and a clear understanding of the approach to employ, we initiated the foundational project planning: antibiotic production with Streptomyces and developing tools to streamline the production of any antibiotic.

There are several Streptomyces species known for their ability to metabolize antibiotics widely used in the pharmaceutical industry. Guided by the experience of one of our advisors, we contemplated working with the following species:

Both of these species are well-known to our advisors, who possess substantial material and experience in their laboratories. The decision regarding which chassis to select is a challenging one due to the expertise of our advisors, and we remain uncertain about the most suitable choice.

The choice of chassis is a critical decision in any synthetic biology (synbio) project. It serves as the foundation where your circuit will function and where you'll conduct your experiments. Using actinobacteria in a synbio project comes with unique challenges and intricacies, particularly due to its relatively long life cycle compared to other bacteria. Additionally, optimal growth conditions are essential to ensure that sufficient material can be collected for testing within a reasonable time frame for competition.

After extensive meetings and diligent research, we grappled with the decision between two options, realizing the necessity for a species that produces well-known antibiotics and is reliable for testing. We had to select the most common species among them.

For six months, our primary chassis was Streptomyces venezuelae. Our entire visual identity and research efforts revolved around this species. We delved into the metabolic pathway of chloramphenicol and the development of this bacterium. However, a significant challenge emerged when we considered conducting experiments. This particular species was not readily available in Brazil, necessitating its importation from Germany. This posed a substantial problem for us, as we couldn't secure the necessary funding or afford the time required to import it. Thus, we had to explore our alternative option, a Brazilian species that was readily accessible and had brought us to this juncture.

Nonetheless, this change was not a setback for our team. Upon making this shift, we restructured our team to work with Streptomyces olindensis and Cosmomycin D. In retrospect, this alteration proved to be the best decision we could have made. Notably, these organisms were already present in our institute, thanks to the generosity of one of our esteemed professors, Gabriel Padilla Maldonado, from the Institute of Biomedical Sciences. This transition opened new avenues for our project. We discovered that Cosmomycin D, in addition to its antibiotic properties, had potential as an antitumoral agent [5]. This revelation expanded our project's scope, as we aimed not only to combat super bacteria but also to contribute to cancer treatments. This was particularly significant, as it kindled a spark of hope, coupled with the fact that we had the species readily available in our university.

To adapt to this change, we embarked on a journey to revisit our research, uncover the genomic clusters, and decipher the metabolic pathways responsible for Cosmomycin D production representated by figure 2. We delved deep into understanding the intricate mechanisms that underlie our product. This effort allowed us to amass a wealth of knowledge, enabling us to perform comprehensive testing and seek ways to generalize our approach, making it applicable to various Streptomyces species and antibiotics.

When we first pondered this question, our initial inclination was to modify the culture medium. However, our Principal Investigator brought us back to reality, reminding us that this was not a realm of synthetic biology. This was indeed a valid point, and after extensive deliberation, we identified two primary methods to optimize production:

The construction of both these constructs was relatively straightforward. We already had the genome sequence of Streptomyces olindensis at our disposal. For the Flux Bomb expression, we chose a cloning approach to reduce complexity, while for the plasmid for malonate, we decided to synthesize it. This combination of methods was advantageous for optimization and time efficiency.

The construction process turned out to be more complex and expensive than initially anticipated. Actinobacteria have a high percentage of GC content [1], making our plasmid significantly more intricate than any enterprise could synthesize. Consequently, we focused on cloning and hoped that everything would align as planned.

Both of these ideas were inspired by our advisor, Leandro. We discovered the vital role of Streptomyces Antibiotic Regulatory Proteins (SARPs), a family of regulatory proteins widely distributed across actinobacteria, in regulating antibiotic production. After this realization, our project transitioned from the theoretical realm to practical implementation. We were well-prepared to begin testing our designs and constructs in the laboratory, marking a crucial phase in our project's development.

Our modeling efforts were divided into two main focuses: molecular docking of SARP interactions with promoter regions and the analysis of a mathematical model for metabolic production. We conducted a comparative study involving the wild type, our mutant, and a model described in the literature.

Our initial modeling endeavor aimed to understand the molecular structure of SARPs and their interactions with various promoter sites. We pursued this by employing comparative modeling techniques, leveraging optimized structures from high-performance prediction servers. The HDOCK program played a pivotal role in generating the most favorable validation scores for SARPs across various promoter sites. This approach held substantial promise as it provided valuable insights into how SARPs interact with promoters, shedding light on the mechanisms behind antibiotic production.

To address the metabolic production aspect, we relied on a model developed by Professor Ana Katerine LOBATO, Ana Katerine de Carvalho Lima. Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20. 2010. 209 f. Tese (Doutorado em Pesquisa e Desenvolvimento de Tecnologias Regionais) - Universidade Federal do Rio Grande do Norte, Natal, 2010. during her PhD thesis. Her model encompassed the entirety of our requirements. We integrated her mathematical model, as described on our model page, into our project. Professor Katerine's parameters became our reference, and we aspired to achieve these ideal parameters. Her model had two additional inputs: the concentration of Cosmomycin D produced by wild-type Streptomyces and the concentration produced by our mutant strain.

However, a significant challenge emerged with this approach. Our heavy reliance on data obtained from wet lab tests posed a considerable obstacle, as the progress in these experiments was notably slow, leading to significant delays. As previously me ntioned, the complexity of our genes, along with their high GC content, resulted in a time-consuming gene synthesis process. Consequently, these delays reached a point where we couldn't incorporate the outcomes into our mathematical model within the required timeframe for the wiki freeze. This setback had a substantial impact on the advancement of our project. A comprehensive account of the entire process can be found on our notebook page.

While a substantial portion of our experiments faced delays due to the organism change and the scarcity of our plasmid, additional challenges are elaborated upon in our notebook page. We responded to these issues by carefully managing our supplies and resources.

Our analysis of the modeling tests has provided valuable insights into our project's operation and behavior.

Through protein molecular docking, we successfully predicted all the interaction sites of SARPs with promoters. This knowledge is immensely beneficial, as it allows us to understand how SARPs interact with various conservative domains. This, in turn, equips us with the best approaches to utilize SARPs in different scenarios and apply these processes to the production of various antibiotics by Streptomyces.

Moreover, our findings offer valuable insights to other iGEM teams, helping them understand the expected production levels of secondary metabolites by Streptomyces. We believe that these calculations are crucial for comprehending the metabolic pathways involved in the production of any anthracycline by our bacteria. Reviewing the results of Professor Lobato, as presented on our model page, we have concluded that in simulations, a mutant strain produces more than twice the amount of antibiotics compared to a wild-type strain. This finding aligns with our initial hypothesis and has significant implications for our project.

Our experiments, revealed a multitude of challenges associated with working with Streptomyces, as previously mentioned. The transformation process with Streptomyces proved to be exceptionally challenging, compounded by the issues outlined before, both in this report and in our notebook page.

The Streptomyces strains we worked with exhibited conspicuous red pigmentation, which led us to hypothesize that they were producing Cosmomycin D. However, due to our inability to quantify this production, our understanding remained speculative. Fortunately, Professor Padilha provided us with mutant Streptomyces strains that were known to produce Cosmomycin D.

Notably, our observation revealed that the Cosmomycin D produced by the wild type strains exhibited slightly less red coloration than that produced by the mutants provided by Professor Padilha's students. We are grateful that their work ran concurrently with ours, indirectly aiding our process. Their research into secondary metabolite production was fortunate for us as they successfully cloned Cos I and Cos J, offering us insights into what might occur in future experiments we plan to conduct for presentation in Paris.

Their results indicated a higher production of Cosmomycin D, as their culture medium appeared noticeably redder than our wild-type strains. This served as corroborative evidence and led us to hypothesize that our construct might produce approximately twice as much as their construct. This hypothesis aligns with the mathematical model proposed by Professor Lobato, which suggests our construct has twice the capacity to expel Cosmomycin D from the inner cell region. This data reinforces our initial hypothesis and suggests that through further experimentation, we can achieve increased production.

Drawing from the results of our preliminary tests, we can better analyze them by addressing key questions:

What Went Wrong, and How Can We Fix It?

To enhance our project, we plan to delve into the existing literature and consult with specialists to uncover possible reasons for any shortcomings and devise solutions. While our preliminary results (from modeling and wet lab tests) suggest that our circuit is functioning as intended, some level of uncertainty exists due to the necessity to test on a construct similar but not identical to our own. These promising findings must be validated by further experiments.

Our successful efforts have demonstrated that Streptomyces have the capability to produce substantial quantities of antibiotics, with high potential for use with any anthracycline antibiotic. Additionally, the Eflux Bomb concept shows promise with other antibiotics, given that it effectively removes toxic compounds from the cell, thereby enhancing production and survival in culture medium.

A significant question arises concerning the activity of SARPs themselves. Therefore, for our future experiments, which we plan to present in Paris, we intend to:

With the opportunity to conduct these tests, we aim to further enhance our project by comprehending the reasons behind any setbacks, devising solutions for them, and adhering to the engineering cycle to maintain continuous improvement.