iGEM 2023 — Team usp-brazil

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

![wave](https://static.igem.wiki/teams/4590/wiki/wave3.svg)

In the beginning, we planned three approaches to investigate activation of cryptic genes and how to increase the production of the metabolites under its control.

First, Streptomyces genus is known by its ability to produce numerous secondary metabolites of biotechnological interest, specially for the pharmaceutical industry. The synthesis of these molecules depends on a gene cluster controlled by a specific transcriptional regulator named SARP (Streptomyces Antibiotic Regulatory Protein), which is activated under determined conditions, such as incubation in presence of stressors agents. Unfortunally, despite this genus of bacteria have many gene cluster encoding for putative molecules, most of them can’t be activated, once we don’t know the condition inducing for SARP production. Nevertheless, these proteins are positive transcriptional regulators, which makes it possible to genetically engeener Streptomyces to produce the SARP of interest and, consequently, transcribe the proteins that synthethise the molecule of interest. For this part of the project, we planned to clone the SARP that controls the synthesis of cosmomycin, a antimicrobial and antitumor molecule produced by Streptomyces olindensis, a brazilian isolate, and investigate if it starts to be synthethised in commun culture medium. Our construction would be created by inserting SARP gene in pSET152Ec constitutive vector, digesting with NotI and EcoRI restriction enzymes and ligated with T4 ligase. We designed the primers with the following nucleotide sequences:

Forward 5’-TATAT**GCGGCCGC**GCGATCTATGGGGGAACG-3’

Reverse 5’-TATAT**CTTAAG**TCAGTAGACCCGCACCG-3’

Oligos were synthethized by IDT (Integrated DNA Technology). Curiously, our primers were delivered twice to somewhere in Florida before sending them to us. Once we received it, PCR was prepared using the high fidelity Phusion polymerase from Thermo Scientific with the following protocol:

30.5 µL H2O; 1 µL dNTPs; 1.5 µL DMSO; 10 µL 5x GC buffer; 2.5 µL primer foward in 10 µM concentration; 2.5 µL primer reverse in 10 µM concentration; 1.5 µL DNA template and 0.5 µL enzyme.

The reaction was incubated as:

30s at 98°C

10s at 98°C\*

30s at 61°C\*

60s at 72°C\*

5 min at 72°C

hold at 4°C

34 cycles repeating the steps indicated by \*

Streptomyces colonies were boiled at 95°C for 10 min and used as DNA template for the first two reactions, which yielded significant low amounts of amplicon, impossible to use for digestion and ligation reactions

![Image 1](https://static.igem.wiki/teams/4590/wiki/colony1.svg)

![Image 2](https://static.igem.wiki/teams/4590/wiki/colony2.svg)

For the next reaction, instead of the whole colony, we used genomic DNA extracted from Streptomyces using the Wizard genomic DNA purification kit from Promega. We have a lot of trouble with this protocol, since the mycelium formed during the bacterial growth makes the lysis and DNA purification difficult. After three trials, we got enough DNA for PCR, the reaction was prepared with the same protocol as described above, but with a gradient of temperatures ranging from 57 to 68°C (57, 59, 62, 65, 68°C). As showed bellow, the best reaction occured at 68°C where the unspecific band above the desired one wasn’t present. Inserts were purifed from agarose gel using Thermo Scientific GeneGET Gel extraction kit and stored at -20°C until the digestion reaction.

![Image 3](https://static.igem.wiki/teams/4590/wiki/colony3.svg)

For the next step, we attempted to obtain a higher amounts of pSET152Ec transforming chemocompetent DH5α bacteria by heat shock, incubating overnight at 37°C in 2xTY medium supplemented with 50 μg/mL apramycin, using vector and antibiotic that was given to us. For our surprise, there was a background population of bacteria growing in the agar, which made us believe the antibiotic wasn't effectively selecting the transformants. We tried again to transform it now using 100 μg/mL apramycin, but again the same phenotype was seen. Preparing a serial dilution of the antibiotic in microplates, we determined that the ideal concentration would be 200 μg/mL, that in fact selected the positive transformants without the background growth. Two colonies were picked and inoculated in 2xTY medium with 200 μg/mL apramycin and incubated overnight at 37°C under agitation. In the next morning, the plasmid extraction was done and, unexpectedly, it formed a intense 2.000 bp band in the agarose gel, that migrates to 4.000 bp after digestion with NotI enzyme.

![Image 4](https://static.igem.wiki/teams/4590/wiki/colony4.svg)

![Image 5](https://static.igem.wiki/teams/4590/wiki/colony5.svg)

Our vector should have about 5.900 bp, sugesting it was not the expected plasmid. Bacteria was transformed and DNA extracted again, revealing the same result. A new aliquot was given to us, the protocol was repeated again and now the plasmid extraction yielded a construct with 4.000 bp that migrates in the gel forming a band between 7.000 and 8.000 bp after digestion with NotI enzyme. The size diverges a bit with the expected one, but we thought in the possibility that something was already cloned in this plasmid, so we could substitute the insert with our SARP amplicon when digesting with the two described enzymes. Plasmid and insert digestions was prepared with NotI and EcoRI enzymes, and ligation with T4 ligase. DH5α was transformed with 10 ng of DNA from ligation, selected by apramycin, and using a control reaction containing only the plasmid digested and ligated, without insert. For our surprise, the control and the reaction had the same number of colonies, indicating the reaction didn't was not successful. We prepared a new digestion, ligation and transformation, but the same result was obtained. To check if the plasmid was being digested as expected, 3 digestion reactions were prepared using both enzymes (NotI and EcoRI) or each enzyme separately. We found that the plasmid was linearized by NotI enzyme, bot not dor EcoRI. Plasmid was purified again but the digestion reveald the same pattern, making it impossible to conclude the cloning.

For the second part, we wanted to explore the possibility of increasing cosmomycin production through the elevated expression of resistance genes. CosI and cosJ genes encoding for the autotransporter responsible for cosmomycin exportion would be cloned in pSET152Ec using the same strategy descibred for SARP. Also, a construction with cosI, cosJ, cosP and cosU would be prepared, the 4 genes related with cosmomycin resistance. Since we couldn't correctly digest the vector, we were not able to clone it also.

The third part was focused on improving the malonate uptake, increasing the intracellular substrate for cosmomycin production and consequently increasing its production. We designed pIJ6021 plasmid transforming it in a conjugative plasmid by adding the sequences recongnized for mobility. Then, once we didn't found a di-carboxylate carrier protein in the genome of Streptomyces olindensis (only a tri-carboxylate carrier protein related to citrate uptake), we inserted the gene of a di/tri-carboxylate carrier protein from Streptomyces hygroscopicus and a malonyl-CoA enzyme from Rhizobium leguminosarum. For this part, instead of cloning, we planned to synthesize the whole plasmid, but we were informed that the wouldn’t be possible to create the whole plasmid. For this reason, we devided the plasmid in two minor fragments that would be ligated using SLIC. unfortunately, the companies couldn’t synthesize this plasmid due to the high G-C content. We asked if it would be possible at least to produce a synthetic sequence of only the two proteins, but the G-C content was too high even for this.

Our construct can be found on: [Primer and Plasmid Constructions](https://docs.google.com/document/d/1x9EjMhq7JPESwp7tXevP6FIsvdw7pWGxKB56ZHJv0xM/edit)

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{% block title %}Engineering{% endblock %}
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# Engineering Success

![wave](https://static.igem.wiki/teams/4590/wiki/wave.svg)

## GLIMPSE INTO OUR JOURNEY

![find-and-understand](https://static.igem.wiki/teams/4590/wiki/find-and-understand.svg)

Figure 1: Representation of our enginering sucess journey

Streptomyces spp. are a remarkable group of microorganisms that have garnered significant attention in the fields of microbiology, biotechnology, and medicine. These filamentous bacteria belong to the Actinobacteria phylum and are renowned for their immense importance in various aspects of life on Earth. Streptomyces species play a pivotal role in the natural environment, the laboratory, and industrial applications, especially in the synthesis of new antibiotics. This makes them a subject of profound interest and study [1].

The development of our project over the last two years can be divided into several steps that form our engineering cycle. The story begins with the conceptualization of Strep-by-Strep. We conducted extensive research, delving into the literature and consulting specialists to comprehend the problem and explore potential solutions.

The entire process was challenging, and it forged a special bond among our team members, helping us grow and become more mature and prepared for the scientific journey ahead. While many of our initial plans faced setbacks, requiring us to restart the process multiple times, we persevered and validated our ideas through modeling and extensive wet lab testing. However, to gain a deeper understanding of our project's operation, we recognize the need for additional experiments in the future to continue and continuously improve the cycle.

The entire process was challenging, and it forged a special bond among our team members, helping us grow and become more mature and prepared for the scientific journey ahead. While many of our initial plans faced setbacks, requiring us to restart the process multiple times, we persevered and validated our ideas through modeling and extensive wet lab testing. However, to gain a deeper understanding of our project's operation, we recognize the need for additional experiments in the future to continue and continuously improve the cycle.

![wave](https://static.igem.wiki/teams/4590/wiki/wave3.svg)

# IDEALIZING AND CREATING STREP-BY-STREP.

## 1st St(r)ep. Find and understand the problem

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.**

## 2nd St(r)ep. Understanding the solution

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:

* **Streptomyces venezuelae:** This bacterial species, belonging to the genus Streptomyces, is a soil-dwelling filamentous bacterium renowned for its remarkable capability to produce various biologically active compounds, especially antibiotics. S. venezuelae was initially isolated from soil samples in Venezuela and has become a significant focus of research due to its capacity to synthesize essential secondary metabolites. One of its most notable achievements is the production of chloramphenicol, a broad-spectrum antibiotic with extensive clinical applications. The life cycle of this bacterium is intricate and includes the formation of mycelia and spore-bearing structures. The sequencing and analysis of the S. venezuelae genome have provided valuable insights into its metabolic potential and antibiotic production pathways. The study of this bacterium has significantly contributed to our understanding of antibiotic biosynthesis and the development of new antibiotics for medical use [2, 3].
* **Streptomyces olindensis:** Another member of the Streptomyces genus, this soil-dwelling bacterium, was initially isolated from soil samples in Brazil [4]. Although it may not be as extensively studied as some other Streptomyces species, S. olindensis holds significant potential for the discovery of novel bioactive compounds, including antibiotics. Similar to other Streptomyces species, S. olindensis exhibits a complex life cycle involving mycelia and spore-bearing structures. Its genetic and metabolic capabilities make it a promising candidate for research into antibiotic production, particularly Cosmomycin D, and other biotechnological applications. While the specific compounds synthesized by S. olindensis and their potential applications remain subjects of ongoing research, it represents a valuable resource in the quest for new antibiotics and biotechnological innovations [5].

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.

## What Should Be Our Chassis?

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.

* **First Choice: Streptomyces venezuelae**

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.

* **Final Choice: Streptomyces olindensis**

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.

![biosynthesis-cluster](https://static.igem.wiki/teams/4590/wiki/biosynthesis-cluster.png)
Figure 2: biosynthesis cluster of cosmomycin D, respectivaly up to down, left to right: Minimum PKS; Aglycone; Glycosyltransferase; Biosynthesis/Sugar modification; Aglycone modification; Unknow function; Regulator; Resistence. The arrow direction represents the sense of transcription

### How Can We Optimize Production?

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:

* **Synthesis of Malonate:** Our first approach involved producing a fatty acid precursor. It's crucial to note that the antracyclin class of organic compounds, of which Cosmomycin D is a member , follows a metabolic pathway that utilizes fatty acids as raw materials to yield the final secondary metabolite. Hence, by enhancing the production of the rate-limiting reaction in this pathway, we aimed to boost the final product's production [6, 7, 8].
* **Flux Bomb Superexpression:** The second method we considered was to superexpress the flux bomb. By doing so, we aimed to facilitate the expulsion of Cosmomycin D, a compound that is toxic to Streptomyces, and increase its excretion. This would ensure that Cosmomycin D is continuously expressed without reaching levels that could shut down the secondary metabolite's production pathway [6, 9].

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.

![plasmid-construction](https://static.igem.wiki/teams/4590/wiki/plasmid-construction.png)
Figure 3: Plasmid construction for synthesis of malonate

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.

![wave](https://static.igem.wiki/teams/4590/wiki/wave6.svg)

# TESTING STREP-BY-STREP

## 1. Modeling Tests

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.

### Molecular Docking

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.

### Metabolic Production Model

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D), 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D).

## 2. Wet Lab tests

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D). We responded to these issues by carefully managing our supplies and resources.

# LEARNING FROM THE RESULTS

# MODELLING RESULTS

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D), 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.

![wave](https://static.igem.wiki/teams/4590/wiki/wave4.svg)

# WET LAB RESULTS

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.

## In the Future

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.

## New 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:

* **Continue experiments with our constructs:** We anticipate that improved expression will result in significant advances in the quantification of antibiotic production. These experiments will help confirm and expand upon the results obtained with Professor Padilha's construct, allowing us to draw conclusions about what aspects of our design have been effective.
* **Explore SARP interactions:** We aim to create two plasmids, one for SARP production and another that only activates when SARPs interact with their respective promoters, triggering the expression of GFP. This approach will provide a better understanding of SARP interactions with various promoters, offering insights into which promoters exhibit greater activity.
* **Utilize HPLC and MS (High-Performance Liquid Chromatography and Mass Spectrometry):** These techniques will allow us to analyze and quantify the precise amount of Cosmomycin D present, enabling us to determine the exact quantity produced. This data will provide insights into the potential market benefits, as we'll know precisely how much more we've produced.

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.

## References:

[1] DE LIMA PROCÓPIO, R. E. et al. Antibiotics produced by Streptomyces. **The Brazilian Journal of Infectious Diseases**, v. 16, n. 5, p. 466–471, 1 set. 2012.

[2] POGUE, J. M. et al. 146 - Tetracyclines and Chloramphenicol. In: COHEN, J.; POWDERLY, W. G.; OPAL, S. M. (Eds.). **Infectious Diseases** (Fourth Edition). [s.l.] Elsevier, 2017. p. 1256-1260.e1.

[3] KIM, E. J.; YANG, I.; YOON, Y. J. Developing Streptomyces venezuelae as a cell factory for the production of small molecules used in drug discovery. **Archives of Pharmacal Research**, v. 38, n. 9, p. 1606–1616, 1 set. 2015.

[4] ROJAS, J. D. et al. Genome Sequence of Streptomyces olindensis DAUFPE 5622, Producer of the Antitumoral Anthracycline Cosmomycin D. **Genome Announcements**, v. 2, n. 3, p. e00541-14, 26 jun. 2014.

[5] CASTILLO ARTEAGA, R. D. et al. Mycothiol Peroxidase Activity as a Part of the Self-Resistance Mechanisms against the Antitumor Antibiotic Cosmomycin D. **Microbiology Spectrum**, v. 10, n. 3, p. e0049322, 29 jun. 2022.

[6] 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.

[7] CHEN, A.; RE, R. N.; BURKART, M. D. Type II fatty acid and polyketide synthases: deciphering protein–protein and protein–substrate interactions. **Natural Product Reports**, v. 35, n. 10, p. 1029–1045, 17 out. 2018.

[8] OLANO, C. et al. Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. **Metabolic Engineering**, v. 10, n. 5, p. 281–292, 1 set. 2008.

[9] Kong D, Wang X, Nie J and Niu G (2019) **Regulation of Antibiotic Production by Signaling Molecules in Streptomyces**. Front. Microbiol. 10:2927. doi: 10.3389/fmicb.2019.02927

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

![wave](https://static.igem.wiki/teams/4590/wiki/wave3.svg)

All experiments conducted were based on classical techniques in the fields of molecular and synthetic biology, as detailed in the protocol and notebook sections.

## PCR

The initial experiment involved PCR to amplify the genetic material obtained from the synthesis carried out by IDT. This amplification was necessary to enable subsequent plasmid digestion. PCR was also employed in conjunction with agarose gels to confirm amplification and identify the genomes of the bacterial strains used.

## Plasmid Digestion

To proceed with the experiments, the already amplified primers and the restriction enzymes NotI and EcoRI were used to insert the SARP gene into pSET152Ec, which exhibits constitutive expression. However, several combinations with these enzymes were required to achieve the correct model with the expected molecular weight.

## Transformation of DH5α

After confirming the digestion of the plasmid, we attempted to transform DH5α bacteria to augment the quantity of available plasmids. Unfortunately, we encountered several challenges during this process, including difficulties in confirming the vector's digestion, ensuring the successful transformation of the bacteria, and facing challenges in selecting antibiotic-resistant strains from the medium. Consequently, we were unable to test the hypothesis regarding SARP regulation and the modulation necessary to enhance cosmomycin production.

## Metabolic Engineering

Our experiments aimed to reconfigure the metabolic pathways of the bacteria to boost cosmomycin production by increasing the availability of malonate. Initially, we planned to synthesize a specific plasmid for this purpose. However, due to its high G-C content and size, synthesizing the plasmid proved unfeasible, even after attempting to divide it into two fragments for later assembly. Furthermore, the same issue of a high G-C base composition prevented us from producing the synthetic enzyme needed to enhance malonate production.

## Used Protocols:

### PCR

PCR was prepared using the high fidelity Phusion polymerase from Thermo Scientific with the following protocol:

Primer Forward 5’-TATAT**GCGGCCGC**GCGATCTATGGGGGAACG-3’

Primer Reverse 5’-TATAT**CTTAAGTCAG**TAGACCCGCACCG-3’

30.5 µL H2O; 1 µL dNTPs; 1.5 µL DMSO; 10 µL 5x GC buffer; 2.5 µL primer foward in 10 µM concentration; 2.5 µL primer reverse in 10 µM concentration; 1.5 µL DNA template and 0.5 µL enzyme.

The reaction was incubated as:

30s at 98°C

10s at 98°C\*

30s at 61°C\*

60s at 72°C\*

5 min at 72°C

hold at 4°C

34 cycles repeating the steps indicated by \*

## Digestion and Ligation of the plasmid and Transformation of bacteria

### Materials:

* Plasmid DNA
* Restriction enzymes NotI and EcoRI
* T4 DNA Ligase
* Incubator or Water Bath (37°C)
* Heat Block or Thermocycler (65°C)
* Gel Electrophoresis Equipment
* Agarose Gel
* DNA Size Marker
* Gel Extraction Kit

### Protocol:

#### 1. Digestion of Plasmid DNA and DNA Fragment(s)

1.1 Set up a master mix for each digestion reaction as follows (adjust volumes as needed):

* 1. Plasmid DNA
* Restriction enzymes NotI and EcoRI
* T4 DNA Ligase

1.2. Mix gently by pipetting and centrifuge briefly to collect all liquid at the bottom of the tube.

1.3. Incubate the reaction mixtures in a 37°C incubator or water bath for the recommended time (usually 1-2 hours). Ensure that the reaction tubes are securely capped to avoid evaporation.

1.4. After digestion, inactivate the enzymes by heating the reaction mixtures at 65°C for 10-15 minutes.

#### 2. Analyze Digestion

2.1. Perform agarose gel electrophoresis to confirm the success of the digestion. Should have distinct bands corresponding to the expected sizes.

#### 3. Purify Digested DNA Fragments

3.1. The purification of DNA was done using Thermo Scientific GeneGET Gel extraction kit

#### 4. Transformation of bacteria by heat shock

4.1. With a pellet of DH5α, ressuspend the bacteria in a solution of calcium chloride and incube for 30 minutes;

#### 5. Plating and Selection

5.1. Plate the transformed cells on selective agar plates containing the appropriate antibiotic(s) to select for the desired plasmid.

#### 6. Incubation and Analysis

6.1. Incubate the plates at the appropriate temperature for the host organism until colonies appear.

6.2. Analyze with a agarose gel electrophoresis the colonies by plasmid isolation to confirm the presence of the desired insert.

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# Results

![wave](https://static.igem.wiki/teams/4590/wiki/wave3.svg)

## Dry-Lab

### Model Metabolic

The work titled "METABOLIC FLUX ANALYSIS FOR OPTIMIZING THE SYNTHESIS OF COSMOMYCIN ANTIBIOTIC BY STREPTOMYCES OLINDENSIS ICB20" (LIMA LOBATO, A. K. C., 2010) demonstrated that the production of cosmomycin can be computationally estimated with good experimental reproducibility and highlights the correlation between the fatty acid synthesis pathway and the cosmomycin pathway. This supports the hypothesis that the incorporation of malonate as a precursor of Acetyl-CoA would increase the production of cosmomycin without compromising an essential pathway for the microorganism's survival.

#### Model Protein and validation

Using alphafold, we predicted five potential structures for our SARP protein. Then, we did a simple validation of the protein structure using SWISS-MODEL, that displays ligands, global and local quality, and target-template alignments. The third of the five structures we predicated was the one with the best outcomes.

![Validation](https://static.igem.wiki/teams/4590/wiki/validation.svg)

Figure 1. Validation of modeled protein input number 3 using Ramachandran plot of SWISS-MODEL analysis

| Input | Mol Prob | Clash Score | Ramach Fav | QMEANDisCo |
| --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 |
| 2.16 | 2.16 | 1.99 | 2.19 | 2.17 |
| 17.28 | 17.05 | 11.82 | 18.64 | 17.51 |
| 93.53% | 93.53% | 93.88% | 93,53% | 93,53% |
| 0.70 | 0.70 | 0.71 | 0.70 | 0.70 |

Table 1. Results of all generated structures. The inputs 1 through 5 correspond to the built structures in Alphafold. Additionally, we have scores produced by MolProbity, ClashScore, Ramachandran Favoured, and QMEANDisCo Global. The third structure was the one that gave the best results.

It is possible to assess the precision of the predicted protein structure using the Ramachandran plot. A positive indication that our protein's structure is close to the genuine conformation is that our Ramachandran plot values were above 90%. MolProbity creates a single number by combining all geometric scores (such as clashscore, bad bonds, bad angles) to indicate quality. Higher percentiles and lower values are preferable. Finally, we have the QMEANDisCo parameter, a single model approach that combines distance constraints (DisCo) score, statistical potentials, and agreement terms. DisCo assesses the consistency of pairwise CA-CA distances obtained from a model using homologous structure-extracted constraints.

![QMEANDisCo score of protein number 3](https://static.igem.wiki/teams/4590/wiki/qmeandisco-2.svg)

Figure 2. QMEANDisCo score of protein number 3

The SARP family has two conserved domains: the DNA-binding transcriptional activator of the SARP family (DnrI) and the Bacterial Transcriptional Activation domain (BTAD). By aligning these domains with the sequence of the Cosmomicin SARP, we were able to identify the position where these domains are located in the predicted structure.

![Structure of the SARP](https://static.igem.wiki/teams/4590/wiki/sarpcolagem-1-2.svg)

Figure 3: Structure of the SARP rotated by 90 degrees. In a) The SARP, in b) we have the DnrI domain highlighted, in c) we have the BTAD domain highlighted, and in d) we have both the DnrI and BTAD domains marked.

As expected, the conserved domains that we know from the literature interact with DNA are located within the protein's cavity, indicating that it is indeed the interaction site with DNA.

## Wet Lab

### Transformation Challenges

The transformation process with Streptomyces proved to be exceptionally challenging. This was further compounded by the issues outlined throughout our research and in our project notebook. Despite these obstacles, our team persevered in our quest to optimize antibiotic production.

### Pigmentation Clues

One of the intriguing observations during our experiments was the conspicuous red pigmentation exhibited by the Streptomyces strains we worked with. This distinctive coloration led us to hypothesize that these strains were potentially producing Cosmomycin D, an important secondary metabolite of interest. However, quantifying this production proved to be a significant challenge, leaving our understanding largely speculative.

### Collaboration and Insights

To advance our understanding of Cosmomycin D production, we were fortunate to receive support from Professor Padilha. He provided us with mutant Streptomyces strains known to produce Cosmomycin D. This collaboration greatly enriched our research efforts.

Notably, our observations revealed an intriguing difference in the red pigmentation between the Cosmomycin D produced by the wild-type strains we initially worked with and that produced by the mutant strains provided by Professor Padilha's students. The mutant strains exhibited a richer red coloration. This unexpected discovery suggested that the mutants produced a higher quantity of Cosmomycin D.

### Building on Insights

The parallel research conducted by Professor Padilha's students, focusing on secondary metabolite production, was fortunate for our project. They successfully cloned Cos I and Cos J, providing valuable insights into future experiments.

Our observations and data analysis indicated a notably higher production of Cosmomycin D in the mutants provided by Professor Padilha's students. The culture medium used by these mutants appeared significantly redder than that of our wild-type strains. This corroborative evidence reinforced our initial hypothesis.

Furthermore, we speculated that our construct had the potential to produce approximately twice as much Cosmomycin D compared to the wild-type strains. This hypothesis aligned with a mathematical model proposed by Professor Lobato, which indicated that our construct could expel Cosmomycin D from the inner cell region at twice the rate of the wild-type strains.

### Future Directions

These findings and insights have set the stage for future experimentation. Our aim is to continue exploring and optimizing the production of Cosmomycin D in Streptomyces. Through further research, we are confident that we can achieve increased production of this important secondary metabolite.

Our collaborative efforts, innovative approach, and dedication to advancing antibiotic production in Streptomyces serve as a testament to our commitment to this vital field of research.

**Reference**

Studer, G., Rempfer, C., Waterhouse, A.M., Gumienny, G., Haas, J., Schwede, T. QMEANDisCo - distance constraints applied on model quality estimation. Bioinformatics 36, 1765-1771 (2020).

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