Week 1 HW: Principles and Practices

Important: I use ChatGPT and Gemini to help me organize my ideas, translate some concepts and reunite everything!!

Chagas disease is a major public health problem in Latin America, especially in underserved regions. In Argentina, this disease mainly affects the northern part of the country, where high temperatures, rural housing conditions, poverty and limited access to healthcare favor the presence and spread of the insect vector vinchuca or “kissing bug” (scientifically called Triatoma Infestans). Many people living in these areas are diagnosed late or not diagnosed at all.

Early diagnosis is extremely important, since treatment is much more effective in the early stages of infection. However, current diagnostic methods often require multiple tests, specialized equipment, and trained personnel, which are not available at all in low-resource settings. In addition to this, several provinces such as Santiago del Estero, Formosa, Chaco, Tucuman and Jujuy include regions that are extremely difficult to access. Areas like El Impenetrable in Chaco or the rural forested regions are home of communities that lack basic services such as water, electricity and reliable transportation. These create favorable conditions for the presence and reproduction of vinchucas.

Given this, I propose the development of a nanobiosensor for the early diagnosis of Trypanosoma cruzi infection. The idea is to create a portable, low-cost diagnostic tool that could detect parasite-specific biomarkers such as Tc24 and SAPA proteins in simple biological samples like blood, allowing the fast and early diagnosis outside of centralized laboratories.

Governance/Policy goals

The main governance goal of this project is to ensure that the nanobiosensor is developed and used in a safe, ethical and socially responsible way, and it’s accessible to everyone, in order to maximize its public health benefits and minimizing potential harms.

1.  Prevent harm and ensure safe use (non-malfeasance): this technology should not cause any harm due to inaccurate results, misuse, or unsafe handling. It should be easy to use, handled by a primary care doctor.
   

2.  Promote equity and fair access: this nanobiosensor should be accessible to everyone, especially to those most affected rural communities. The goal is the implementation of “Open Science” licensing for sensor’s design to allow local manufacturing in Argentina, reducing dependency on expensive imports, to avoid creating a technology that only benefits massive healthcare systems.
   

3.  Build trust and responsible data use: people must trust the diagnostic tool and the institutions using it. This includes clear communication, informed consent, responsible use of data, and education in healthcare systems and machinery.
   

Potential Governance Actions

a. Field-based validation and approval for point-of-care diagnostics

•    Purpose: nowadays, the diagnosis of Trypanosoma cruzi infection relies mainly on laboratory-based methods such as ELISA and PCR, which require specialized infrastructure and trained personnel, limiting their use in rural and low-resource endemic areas. At the same time, recent studies have explored biosensor-based diagnostics for other parasitic diseases, including malaria and leishmaniasis, demonstrating the potential of nanobiosensors for rapid and point-of-care detection. However, similar technologies for Chagas disease remain underdeveloped and poorly implemented.
  

•    Design: this biosensor would be tested in real environmental conditions (high temperature, limited infrastructure, low-income facilities) in collaboration with local primary care centers and rural doctors. National health authorities (ANMAT) would review the results before approving large-scale use.
  

•    Assumptions: it is expected that the validation and approval would be fast and without obstacles. We could also assume that communities and healthcare professionals will easily adopt this technology but we have to think about a potential resistance from large pharmaceutical companies or major laboratories that may see their market threatened.
  

•    Risks of failure and success:
  

  • Risks of failure: if major corporations oppose the technology, it could lead to delays in approval and, consequently, limited availability. This sensor could also not be well-received due to a lack of local infrastructure or training.
  • Risks of success: If the sensor is successfully adopted, it could create a demand that outpaces local production capacity, potentially causing shortages or uneven distribution.

b. Incentives for affordable and open-access design

•    Purpose: to reduce inequality in access, this action focuses on keeping the technology affordable and widely available.
  

•    Design: public funding or academic grants could require that the nanobiosensor design be shared under open or non-exclusive licenses. Partnerships with public institutions, important researchers and local manufacturers could help reduce costs and support regional production.
  

•    Assumptions: this assumes that researchers and developers are willing to share designs, data and manufacturing processes under open or non-exclusive licenses. It also assumes that open access or publicly funded models can remain economically sustainable while maintaining quality standards.
  

•    Risks of failure and success:
  

  • Risks of failure: if public funding is insufficient or inconsistent, production quality could decline, resulting in unreliable diagnostic devices. Private companies may also be discouraged from participating due to limited commercial incentives.
  • Risks of success: if open-access designs are widely adopted, a lack of clear coordination and quality control could lead to fragmented manufacturing, variable device performance or misuse.

c. Training healthcare workers and engaging communities

•    Purpose: a diagnostic tool is only useful if used correctly and understood by both patients and healthcare workers.
  

•    Design: basic training programs would be implemented for healthcare workers on how to use the sensor, interpret results and explain them to patients. Community education could help reduce fear, stigma or misinformation related to Chagas disease, and also help prevent the infections.
  

•    Assumptions: this assumes that training programs can be effectively delivered, that healthcare workers have sufficient time and institutional support to participate and that communities are open to engaging with new diagnostic technologies and to receiving education.
  

•    Risks of failure and success:
  

  • Risks of failure: without adequate training or follow-up, healthcare workers may misinterpret results or use this tool incorrectly, leading to inaccurate diagnoses or loss of trust.
  • Risks of success: if training and community engagement are highly effective, diagnosis rates may increase rapidly. This could potentially overwhelm healthcare systems that are not fully prepared to provide treatment, monitoring, or long-term care.

Based on the previous analysis, I would recommend a combination of Field validation and approval and Incentives for local manufacturing. I strongly believe that these actions are not sufficient on their own. For example, sharing the sensor’s “blueprints” ensures this technology is accessible and easy to develop, but it does not guarantee the quality control that only formal regulation can provide. Consequently, I propose that ANMAT as a regulatory agency and the Ministry of Health of Argentina should provide funding and legal approval exclusively to projects that commit to manufacturing this nanobiosensors, and keeping prices accessible not only for the public health system, but also for researchers and scientific institutes.

What we win and what we risk:

• To make the sensor low-cost and locally made, we must take on more responsibility in supervising the process. We will be choosing the “harder” path of managing our own production instead of the “easier” but more expensive path of importing finished technology from abroad. It’s the only way to become independent.
• Adjusting and testing the devices in the extreme conditions where most of the damned patients live will delay the official launch. However, this is a necessary sacrifice. Otherwise, we risk the sensors failing due to environmental conditions, leading to false negatives and destroying the community’s trust in the program.

Assumptions and Uncertainties:

• Political will: this plan assumes that the National Health System will keep Chagas disease as a priority and will not cut the budget needed for its treatment.
• Pathogen evolution: it is well known that pathogens tend to mutate in order to adapt and survive new environmental conditions, so it is uncertain if the protein used in this sensor will continue to function in a future. This is why this device will need periodic updates, as well as the studies on T. cruzi.
• Digital infrastructure: we assume that, even in remote areas, there will be a basic way to perform this analysis and measurements, and there will always be a healthcare worker around to do it.

Week 2 HW: DNA Read, Write and Edit

–> This image shows DNA fragments separated by agarose gel electrophoresis and stained with a fluorescent dye, performed during my Genetic Engineering course.

Part 1: Benchling and In-silico Gel Art

According to the instructions, this is the Gel I designed for p53 human protein (tried my best to make it look like a butterfly i’m sorry!!)

Part 3: DNA Design Challenge

For this assignment I have chosen the human protein p53, often called “guardian of the genome” is a critical tumor suppressor that maintains genomic stability by regulating cycle cell processes, promoting DNA repairing, and inducing apoptosis or senescence in response to cellular stress. It acts as a transcription factor, binding to DNA to stop damaged cells proliferation, potentially cancerous genetic material. I find this protein interesting because of its crucial role in maintaining cellular integrity. I was drawn to it during my medical genetics course last year due to its key function in controlling cell proliferation and its ability to trigger cell cycle arrest or apoptosis when DNA damage is detected. This protein is mutated in nearly 50% of known cancers in humans, and it is also involved in processes like aging, metabolism and DNA repair. This wide range of actions makes p53 an incredibly interesting protein to study in synbio.

After doing the reverse translation on my protein sequence to obtain the DNA sequence, I have performed the codon optimization. This is a fundamental part of genetic engineering because different organisms prefer different codons to code for the same amino acid. By optimizing the codons, you align the sequence with the organism’s natural tRNA abundance, which improves translation efficiency. Without doing this, even if the gene is correct, it might not be efficiently expressed, which can lead to low protein yields or even no expression at all. I have chosen the optimization for human organisms and I would prefer to express them on HEK293 cells because they are human-derived, making them ideal for expressing human proteins. There are easy to transfect, grow quickly, and they support post-translational modifications, which are crucial for many human proteins.

Now I have the optimized sequence, to produce the protein from the DNA, it is possible to amplify the sequence using PCR, designing primers that include specific restriction sites. Then, I amplify my gene and insert it into an expression vector such as a pEASY or pUC plasmid. This vector has to contain a strong promoter, like the CMV promoter, which is essential for expression in eukaryotic cells. After that, I have to linearize the plasmid using the same restriction enzymes, and transform the HEK293 cells by electroporation. Then, the cells are incubated in fresh culture medium to allow recovery and expression of the introduced DNA. The plasmid is transcribed into mRNA and then translated into the target protein by the cellular machinery. It is also important to have a selectable marker, such as antibiotic selection, to know which cells are expressing the gene. Finally, protein expression can be confirmed using techniques such as Western Blot, ELISA, or fluorescent detection if a GFP was added.

3.5 (Optional)

A single gene can code for multiple proteins at the transcriptional level primarily through alternative splicing, a process where the pre-mRNA, transcribed from the DNA, is spliced in different ways to include or exclude exons (coding regions).

Alignment of the coding DNA and RNA sequence with its translated amino acid sequence for the p53 protein.

Part 4: Prepare a Twist DNA Synthesis order

I have built the DNA insert sequence according to the instructions on Benchling and this is what I have obtained:

Link to my E. coli sequence: https://benchling.com/s/seq-aEUjDIoXsdjPsD14jzXd?m=slm-BT3BayyvXI3H27cDW11c

Since I could not have access to the Twist Bioscience software (it says I have to contact a distributor), I was not able to continue with this assignment.

Part 5: DNA Read/Write/Edit

5.1 DNA Read

(i) I would like to sequence the DNA of the parasite Tritrichomonas foetus, as it is the microorganism I plan to study for my final project. Specifically, I am interested in identifying the genes encoding excretion-secretion antigens, which are believed to play a key role in host-parasite interactions and reproductive impairment, and I want to probe that. Sequencing its DNA would allow me to better understand the molecular basis of its pathogenicity and how these antigens may affect the reproductive capacity of BALB/c mice. Additionally, obtaining genomic information could help identify potential targets for diagnostics, treatments or preventive strategies against infections caused by this parasite.

(ii) I would use NGS for whole-genome sequencing (WGS), as it provides comprehensive coverage of all genes, including my antigens of interest.

This method belongs to second-generation sequencing technologies because it relies on massively parallel sequencing of millions of DNA fragments simultaneously.

The input would be purified genomic DNA extracted from the parasite, which would then be fragmented, followed by adapter ligation and PCR amplification to generate a sequencing library. During sequencing-by-synthesis (e.g. Illumina platform), fluorescently labeled nucleotides are incorporated into the growing DNA strands, and each base is identified by detecting the emitted fluorescence.

The output consists of millions of short sequence reads that can be assembled bioinformatically to reconstruct the genome and identify genes related to pathogenicity and reproductive effects.

5.2 DNA Write

(i) I would like to synthesize a genetic circuit designed as a biosensor to detect excretion-secretion antigens from T. foetus as mentioned above. Early detection in infected bulls is critical because they can act as asymptomatic carriers and spread the parasite to females and other males, causing infertility, significant economic losses in the cattle industry and environmental consequences, as affected animals contribute to the spread of the parasite in pasturelands and water systems. The synthetic DNA construct would include a sensing module responsive to the parasite antigens already mentioned, a regulatory element, and a reporter gene (such as GFP). This biosensor could potentially be used as a rapid diagnostic tool to identify infected animals before the infection spreads within the herd.

(ii) to synthesize the designed genetic circuit, I would use commercial gene synthesis technology, which allows the production of custom DNA sequences with high accuracy, without the need to assemble fragments manually. The essential steps include designing the desired DNA sequence in silico, chemical synthesis of short oligonucleotides, assembly of these fragments into the full-length construct, and cloning into a plasmid vector for delivery. The resulting DNA can be then amplified and used for downstream applications such as transformation into host cells. This method is scalable and precise, however, limitations may include restrictions on how long the DNA sequence can be, the time it takes to receibe the synthesized construct, and possible difficulties with certain sequences, such as those with very high GC content or many repeated regions.

5.3 DNA Edit

(i) I would be interested in editing the human genome using CRISPR-Cas technology to study numerical abnormalities such as trisomy, specifically those causing early miscarriages, preventing babies from reaching full term, or resulting in reduced life expectancy, like Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13). The goal would be to explore whether removing the extra chromosome in early embryonic stages could restore normal gene dosage and improve developmental outcomes. This type of research could help us better understand the genetic mechanisms underlying severe developmental disorders.

(ii) to perform these DNA edits, I would use CRISPR-Cas9 genome editing technology because it allows precise and targeted modification of specific DNA sequences. This system works by using a guide RNA (sgRNA) designed to match the target DNA region, which directs the Cas9 enzyme to create a double-strand break at that site. The cell’s natural DNA repair mechanisms then repair the break, either by non-homologous end joining or homology-directed repair (which is expected to remove or modify genetic material).

Preparation involves designing specific sgRNAs targeting sequences on the extra chromosome, and delivering the complex Cas9-sgRNAs into cells or embryos, typically using vectors.

Limitations of this method include possible off-target effects, incomplete editing efficiency and mosaicism, where not all cells are edited in the same way.

Week 1 HW: Principles and Practices

Week 3: Lab Automation

1. Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.

   Olsen J.V. et al. Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics (2024) Mol Cell Proteomics 23(7), 100790. DOI 10.1016/j.mcpro.2024.100790

This article describes a fully automated workflow for preparing clinical proteomics samples, from protein digestion to loading peptides into Evotips (disposable, tip-based C18 reversed-phase trap columns), ready for LC-MS/MS analysis. They use the Opentrons OT-2 liquid-handling robot, which controls all preparation steps without manual intervention after the initial loading of reagents. The process combines protein capture through aggregation on magnetic beads with enzymatic digestion and, without centrifugation steps, directly transfers the peptides to Evotips using positive pressure, all programmed through downloadable scripts from the Evosep website. Using this method, up to 192 samples can be processed in parallel in approximately 6 h, which equals to 100 samples/day and eliminates human variability. In tests with HeLa lysates, the workflow identified ~8.000 protein groups and ~130.000 peptides using an 11.5-min gradient on the Orbitrap Astral, demonstrating high sensitivity and reproducibility. It was also applied to 192 plasma samples from patients with metastatic melanoma, revealing clinically relevant protein changes.

2. Write a description about what you intend to do with automation tools for your final project.

For my final project, I want to design a nanobiosensor using metallic nanoparticles (such as gold or silver, maybe (if possible) carbon-based materials) to detect excretion-secretion antigens from the parasite Tritrichomonas foetus, which produces a disease called bovine trichomonosis. Automation tools will help make the experiments more reproducible, faster, and less dependent on manual work. I would use a liquid-handling robot (for example the Opentrons OT-2) to automate repetitive lab tasks, such as preparing nanoparticle solutions, mixing reagents, functionalizing nanoparticles with antibodies or aptamers, performing washing steps, preparing assay plates, etc. this would allow testing many conditions at the same time (for example, different nanoparticle types or concentrations) to find the best sensor design. Automation could also be used to test the biosensor performance, maybe by adding samples and controls to plates, preparing serial dilutions of the target antigen, running multiple detection assays in parallel, measuring signals, etc. that would help evaluate sensitivity and specificity of the sensor more efficiently. I also plan to design simple 3D-printed holders to organize tubes, microplates or sensor chips on the robot deck. If available, the Ginkgo Nebula platform could be used to screen different antibodies or binding molecules to find the one that recognizes the parasite protein with the highest specificity, improving the performance of the biosensor.