Hey everyone!
I’m Jami, an Ecuadorian pharmacy student enrolled at Tor Vergata University in Rome. I’ve been immersed in the medical world since childhood, making me fell in love with genetics and microbiology. My interests have evolved into synthetic biology approaches for drug development, looking forward for the future of biopharmaceuticals.
1.First, describe a biological engineering application or tool you want to develop and why. As a pharmacy student, I have become increasingly interested in how drugs move from an initial idea to clinical use, and how many potential compounds fail long before they could reach patients. Drug development is an expensive and time consuming subject, and an ethically complex process, specifically in early stages. But many drugs are left behind as they fail to show strong enough effects or because the costs for further testing is too high.
Pre-Lecture HW: 1.Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy?
The error rate of polymerase estimates to be made errors once every 10x4–10x5 nucleotides polymerized. This compared to the length of the human genome that’s approximately 3.2 billion base pairs long, that would account up to 32 thousand mutations every time a single cell divided. Biology fixes this gap with multiple systems that check for these errors. Its first system is proofreading; a function found in most polymerases. When they add a wrong base, they recognize this error, backtrack and fix it cutting the wrong base and replacing it. Another way, is mismatch repair (NMR), after polymerase is done, a second group of proteins scan the new DNA for any remaining errors.
I designed a drawing with the website that was made available to us https://opentrons-art.rcdonovan.com/. It made it easier to get used to the way the points would be done inside the plate to create our art. Personally I decided to create a flower, took the exact points for each color, green (sfGFP) and red (mrFP1). I integrated the points to the base code, and customized it for the design to be possible, especially with the high quantity of points and the limit of the 20ul pipette.
PART A: CONCEPTUAL QUESTIONS
1.How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)
2.Why do humans eat beef but do not become a cow, eat fish but do not become fish?
3.Why are there only 20 natural amino acids?
4.Can you make other non-natural amino acids? Design some new amino acids.
Subsections of Homework
Week 1 HW: Principles and Practices
1.First, describe a biological engineering application or tool you want to develop and why.
As a pharmacy student, I have become increasingly interested in how drugs move from an initial idea to clinical use, and how many potential compounds fail long before they could reach patients. Drug development is an expensive and time consuming subject, and an ethically complex process, specifically in early stages. But many drugs are left behind as they fail to show strong enough effects or because the costs for further testing is too high.
This has led me to think whether there could be a more efficient way to evaluate the potential of these compounds before progressing into animal models. To address this challenge, I propose an engineered bacterial screening platform that could act as an early, low cost step in the process, especially for those drugs left behind that could be repurposed. This system could be used to report on the activation or inhibition of specific pathways, giving information on targets and if the drug should be continued to be studied before going to an animal model. It would act as an intermediate testing step making it cost effective and reducing the unnecessary animal testing.
This engineered bacterial screening could also be assembled as a living diagnostic tool for diseases that are difficult to detect using conventional methods or invasive procedures, especially when biomarkers are localized or non-specific. Engineered bacteria have already demonstrated potential as diagnostic tools because they can be programmed to detect biomarkers and respond with measurable outputs. And in this application, the engineered bacteria would work as a living biosensor, temporarily sensing for biomarkers and programs to give a clear response. Both applications would be limited to controlled clinical or research settings , allowing for the exploration through these innovative diagnostics while maintaining the commitment for patient safety.
2.Next, describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals.
Ensure patient safety and non-malfeasance
-Prevent uncontrolled bacterial replication, persistence, or dissemination
-Ensure engineered bacteria can be safely eliminated after use
Enhance biosecurity
-Ensure traceability and accountability in their handling and use
-Prevent repurposing of the technology for harmful or unethical applications
Promote ethical and efficient drug development
-Reduce unnecessary animal testing by strengthening early-stage screening
-Improve early decision making on drug continuation or abandonment
-Avoid discarding potentially beneficial drug candidates
Promote equity and accessibility
-Develop cost-effective screening and diagnostic tools
-Prevent socioeconomic or geographic disparities in access
-Encourage drug repurposing for neglected or rare diseases
Maintain transparency and public trust
-Clearly inform patients and research participants about the use of living biosensors
-Communicate risks, benefits, and limitations transparently
3.Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).
Ensure patient safety and non-malfeasance
Mandatory biosafety and containment standards for all engineered bacteria.
With this action the purpose is to prevent uncontrolled bacterial survival or harmful interactions in human hosts. When talking about genetically modified organisms (GMOs), we already have a comprehensive set of laws for them. Taking this into account, for safety it would be required rigorous assessments and protocols for the authorizations for the idea to start being used.
Enhance biosecurity and prevent misuse
Controlled access and traceability of engineered strains
-Actors: Governments, industry, research institutions
-Implementation:
Centralized strain repositories
Record-keeping of users and experiments
-Failure: Unauthorized use → biosecurity risk
The considerable attention to the control of the strains and the people using it through record keeping would help the prevention of engineered bacteria for unethical or harmful purposes. The idea of a data base is a tool, that already currently exists within EU Biosafety, that provides a unified repository of information on GMOs. We could work with them to create a specific repository for this engineering bacteria and keep records of the labs using them for drug development or as a screening tool.
Maintain transparency and public trust
Public communication campaigns and stakeholder engagement
Educate the public about risks, benefits, and uses of engineered bacteria
There should be a full transparency of the techniques and the developments of the idea. Working with the government, communicating to the public about the project could be an easy way to reach the masses and prevent miscommunication or fear of it. Italian BCH AND EU Cartagena Protocol promote the public access of biosafety information already. following in steps, public engagement programs should be used to maintain trust.
Use of engineered bacteria as a mandatory pre-animal testing step
-Actor: Regulatory agencies, pharmacological industry
-Risk: Over-reliance on bacterial models
Requiring bacterial biosensor validation before animal testing would ensure drugs engage intended pathways and reduce unnecessary animal experiments. Although this step would reduce the unnecessary use of animal models, we are assuming bacteria could be able to have the same mammalian pathways, which could give wrong results.
Promote equity and accessibility
Subsidies and open-access initiatives for bacterial screening and diagnostics
-Actors: Governments, funders, industry, NGOs
-Implementation:
Low-cost bacterial screening platforms
Partnerships to reach underserved regions
-Failure: Technology inaccessible in low-income regions
We could implement funding for low-cost bacterial screening platforms by working with public and private partnerships creating global health initiatives and open drug databases so the technology could be used globally.
Does the option:
1
2
3
4
5
Enhance Biosecurity
2
1
2
2
3
• By preventing incidents
1
1
2
2
3
• By helping respond
2
1
2
2
3
Foster Lab Safety
1
2
2
2
3
• By preventing incident
1
2
2
2
3
• By helping respond
2
2
2
2
3
Protect the environment
1
2
2
2
3
• By preventing incidents
1
2
2
2
3
• By helping respond
2
2
2
2
3
Other considerations
• Minimizing costs and burdens to stakeholders
3
3
2
2
1
• Feasibility?
1
2
1
2
2
• Not impede research
2
2
1
2
1
• Promote constructive applications
2
2
2
1
1
1:weak / 2: moderate / 3:strong
Last, drawing upon this scoring, describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties.
Upon the results of the scoring, the governance actions that should be prioritized are the mandatory biosafety Nand containment standards and have control access and traceability to the engineered bacteria strains. These two goals should be at highest priority as it would be the ones preventing incidents across patient and lab safety and protection. Engineered bacteria although it used for the screening and testing tool, the consequences of failing its containment and possibilities of mutations could be severe. Existing EU Legislation don Italian biosafety measures and frameworks give a sturdy foundation for these actions, making them both feasible steps, ensuring the safeguards in our project and validated containments protocols.
Along with the need to maintain biosafety, is linked the controlled access to the engineered strains, which would be the best method to enhance the biosecurity and prevent any misuse. By maintaining repositories and detailed records of strains and ownerships and experimental uses, gives us the chance to rapidly respond to any incidents. Also, as it’s a regulatory access, the use for unethical purposes is discourage. This action builds on existing databases that share the same goals. As safety and security is assured, regulatory recognition for the engineered bacterial screening as mandatory step could be one that should give attention. This action promotes an ethical drug development by reducing unnecessary animal testing and improving early decisions. While it assumes for similar pathways and results to mammals, it reflects into the 3rs principles and offers a cost-effective way to rescue drugs that have been abandoned. There were a lot of trade offs considered when prioritizing these actions. As strong biosafety and security is needed, it becomes a new financial burden on researchers and overall, the industry. Overall, I think it’s always needed to prioritize the safety and security and ethical efficiency when it comes to creating and advancing engineered biology.
Reflecting on what you learned and did in class this week, outline any ethical concerns that arose, especially any that were new to you. Then propose any governance actions you think might be appropriate to address those issues. This should be included on your class page for this week.
Reflecting from this week’s work and class, I’ve realized that the importance for ethics is really needed, specially for synthetic biology. For the example of engineered bacteria, while the idea has beneficial purposes, the same properties that make them adaptable and programmable, also create concerns about misuse and unintended consequences. This made we more a ware of how closely this innovation is tied to responsibility and oversight overall. Another ethical issues that caught mya attention is reducing animal testing and ensuring patient safety., although using engineered bacteria as a pre animal testing step would minimize the used of animal models, it introduces uncertainties in how well these systems could predict humans’ responses. The ideal of heavily simplified the models leads to further skewed decisions in developments that ultimately affects patients
Public trust and transparency are also really important necessity. New technologies can face resistance and we as scientist should be able to include the people into understanding them and relieve those doubts and fear.
Ethical concerns should always be address, creating appropriated governance actions that create a safe and controlled environment, with clear regulatory limits but also the transparency to the people. All of this combined creates a place where innovation is ensured, while maintaining safety and trust.
Bibliography
-European GMO Authorisation Database (EUGinius). (n.d.). GMO authorisation index. European GMO Initiative for a Unified Database System. Retrieved February 2026, from https://euginius.eu/euginius/pages/authorisation_index.jsf (euginius.eu)
-Cartagena Protocol on Biosafety – Biosafety Clearing-House (BCH). (n.d.). Cartagena Protocol on Biosafety. Secretariat of the Convention on Biological Diversity. Retrieved February 2026, from https://bch.cbd.int/en/ (pmc.ncbi.nlm.nih.gov)
-Pant, A., & Das, B. (2022). Microbiome-based therapeutics: Opportunity and challenges. Progress in Molecular Biology and Translational Science, 191(1), 229–262. https://doi.org/10.1016/bs.pmbts.2022.07.006 (review of microbiome-related therapeutic strategies) (PubMed)
-Zhou, Y. (2022). Engineered bacteria as drug delivery vehicles: Principles and prospects. Frontiers in Bioengineering and Biotechnology (review). Retrieved from https://pmc.ncbi.nlm.nih.gov/articles/PMC11611002/ (review of engineering bacteria for therapeutic delivery) (pmc.ncbi.nlm.nih.gov)
-Kulkarni, V. S., Alagarsamy, V., Solomon, V. R., Jose, P. A., & Murugesan, S. (2023). Drug repurposing: an effective tool in modern drug discovery. Russian Journal of Bioorganic Chemistry, 49, 157–166. Retrieved from https://pmc.ncbi.nlm.nih.gov/articles/PMC9945820/ (overview of drug repurposing approaches) (pmc.ncbi.nlm.nih.gov)
Week 2 HW: DNA- Read, Write and Edit
Pre-Lecture HW:
1.Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the length of the human genome. How does biology deal with that discrepancy?
The error rate of polymerase estimates to be made errors once every 10x4–10x5 nucleotides polymerized. This compared to the length of the human genome that’s approximately 3.2 billion base pairs long, that would account up to 32 thousand mutations every time a single cell divided. Biology fixes this gap with multiple systems that check for these errors. Its first system is proofreading; a function found in most polymerases. When they add a wrong base, they recognize this error, backtrack and fix it cutting the wrong base and replacing it. Another way, is mismatch repair (NMR), after polymerase is done, a second group of proteins scan the new DNA for any remaining errors.
2.How many different ways are there to code (DNA nucleotide code) for an average human protein? In practice what are some of the reasons that all of these different codes don’t work to code for the protein of interest?
Proteins are made of Animo acids, macromolecules that are mostly synthetized by humans. We can synthetize 20 standard Animo acids. They are coded by codons (a group of 3 DNA bps) of which we have 64 possible combinations. As there are more than 1 way to produce the same amino acids, we refer to this code degenerative. Because of this system, in theory, you could swap a codon for another a produce the same amino acids as product and the protein should be the same but there’s several reasons this could fail. Sometimes during the transcription and translation process, things can occur, like ribosomes slowing down to let the protein fold or just certain combination of bps make the mRNA unstable degrading before it can be translated. As the protein synthesis relies of multiples factors the degenerative code isn’t always foul proof.
1.What’s the most commonly used method for oligo synthesis currently?
Currently the most commonly used method for oligo synthesis is by using solid-phase phosphonamidite synthesis. The process adds 1 nucleotide at a time 3’-5’ thought 4 repeating steps.
2.Why is it difficult to make oligos longer than 200nt via direct synthesis?
It is difficult because of the cumulative yield and chemical side reactions. Even is the machine being in optimum state teres still some loss happening with every single base addition. By time u wan to reach 200nt, the majority of the material is going to be failure sequences rather than the target.
3.Why can’t you make a 2000bp gene via direct oligo synthesis?
This isn’t possible because the yield becomes essentially zero, as there would be no way to find target in a solution where practically all is incorrect fragments. Also, the purifications are impossible as it is too big to reliably separate all perfect 2000bps.
What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?
Essential Aminoacids
1.Phenylalanine
2.Valine
3.Threonine
4.Tryptophan
5.Isoleucine
6.Methionine
7.Histidine
8.Arginine
9.Leucine
10.Lysine
When you look at the fact that this lysine contingency was the only fail safe for the dinosaurs, believing that its inability to produce lysine would be able to contain a scape seems a bit redundant and negligent. We all need certain amino acids that we aren’t able to produce, that’s why we make nutritious diets for us, as its abundant in nature. If we can survive without producing it on its own by resourcing to get it throughout our diet, it’s a bit irresponsible to thing this only failsafe was going to work, as we are proof of it not being limiting.
Bibliography
-Drake, J. W., Charlesworth, B., Charlesworth, D., & Crow, J. F. (1998). Rates of Spontaneous Mutation. Genetics, 148(4), 1667–1686. [Referenced via ScienceDirect: “Rates of Spontaneous Mutation” regarding replicative fidelity and evolutionary efficiency].
-Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks
While looking the patterns with the different restriction enzymes, I try to create the top part of a cats siluette, basically just its ears.
I used the patterns of the digestion of enzymes: Pvull, Hindll, kpnl, SalI.
While looking into the possibilites of the use of bacteria as drug delivery systems, I saw the concept of designing bacteria with the capability of secreting anti-inflammatory proteins. I chose Interleukin-10, as its a anti-inflammatory cytokine that usually is secreted by immune cells and has a role in reducing the inflammation in tissues. I used this week assignment as a way to explore the possiblity of engineering bacteria to deliver this therapeutic protein, choosing this protein sequence and optimizing it to E.coli as a trial to see the efficacy of this concept.
In your own words, describe why you need to optimize codon usage. Which organism have you chosen to optimize the codon sequence for and why?
The genetic code is degenerate, which means most aminoacids could be encoded by more than one codon. This makes it possible for organisms to have preferences with codon for the expression of aminoacids, creating a bias. To maximaize the protein production, we need to work according to what the host prefers, improving the trnaslation efficiency and reducing errors. I chose to directly optimized for E.coli, as sequence is more a trial to see if the expression of the protein through out the bacteria is possible. Choosing E.coli simplifies the design and clonign workflow, letting us to conceptially see if the therapeutic delivery is possible.
What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.
Some tehcnologies we could use to optain the protein from the DNA, is cell dependent expression. With this technique the IL-10 gene optimized for E.coli, gets used by the bacterial transcription machinery, producing mRNA that gets bound and translated giving us the end product of IL-10.
I decided to include the pelB signal in the Il-10 seuqnece so the protein when it is translated, it can be directed to the periplasmic space where it can be more easily secreted.
To express IL-1O in E.coli, I designed a expression cassette consisting of a constitutive promoter, a strong RBS, and a start codon. I added a pelB signal sequence that makes possible for the bacteria to secrete the protein. We added a HISX7 TAG, to make it easier to purify the proteins, a stop codon and finally a terminator. We choose clonal genes so it can be delivered as a circular plasmid and direclty transformed into the E.coli, and for vector the pTwist Amp High Copy, that gives us a high plasmid copy number resulting into a strong protein expression.
PART 5: DNA READ/WRITE/EDIT
5.1 READ
-What DNA would you want to sequence (e.g., read) and why?
Even if it isnt strictly related to my project, i would like to sequence genes involved in collagen production and extrecellular matrix stability, specially in patients with EDS. Ehler Danlos Syndrome is a group of inherited connective tissue disorders, with a combined stimated prevelance of at least 1 in 5000, even with it being underdiagnosed, where collagen sequences are mutated leading to misfolded collagen and connective tissue fragility. Because of collagen structure being complex and relying in the triple helix assemblance, small mutations can cause big consequences. Studying genes such as COL1A1, COL1A2, COL3A1 in patients with the disorder, it would give insight into how specific mutaitons lead to misfolded colalgen and connective tissue fragility and give better understanding of genotype/phenotype of variants and a lead to develop targeted therapies.
-What technology or technologies would you use to perform sequencing on your DNA and why?
I think the best choice would be a second generation sequencing, like Ilumina NGS. It’s considered 2nd gen because of the ability of sequencing millions of short dna fragments in parallel and requires the amplification of dna before the sequencing. The input could be be genomic dna extracted from the patients blood, which would have to undergo certain preparation steps. After DNA is extracted, it’s mechanically brokendown into short fragments. Synthetic adapters are lgiated to both ends of each fragments, which allows the binding to flow cells. After the fragments are amplified with pcr and then denatured to a single strand to sequence. For Illumina, the sequencing is done by synthesis. It creates cluster generation by binding the dna fragments into complementary oligos onf the flowcells and thru bridge amplificaiton creating the clusters of identical copies. Labeled nucleotides with a flurescent tag and reversible terminators are added and are incorporeated by polymerase 1 at time per cycle. As each nucleotide has a different signal, we can detect which base is being used each cycle. The output is millions of short dna reads, each giving us the nucleotide sequence and the a quality score of each base. It also makes it possible to identify any variants like deletions or insertions, which woulds allow the detection of any mutation for EDS in collagen genes.
5.2 WRITE
What DNA would you want to synthesize (e.g., write) and why?
I would like to synthesize a correct repair template for the pathogenic mutations in the collagen genes. The many of the mutations associated with EDS, involve the susbtitution of glycine residues in the repeating Gly–X–Y motif that stabilizes the collagen triple helix. If glycine is substituted for a bulkier aminoacid it disrupts the folding of the protein and weakes the connective tissues. If we could synthetize a fragment with the proper repreats and a way to flank the mutation site, it could be used to investigate the possiblities to correct the misfolded collagen and help compared stability of proteins.
What technology or technologies would you use to perform this DNA synthesis and why?
I would use the synthesis thru phosphoramidite chemistry. This methods chemically synthetizes base by base short dna oligonucleotides using protected nucleotides. The overlappping oligos are assembled into a full fragment that its cloned to a plasmid, and later the sequence is verified. This tecnique gives me a high fidelity which is needed for the repeated collagen sequences, and enables precise contorl over mutation correction.
5.3 EDIT
What DNA would you want to edit and why? What technology or technologies would you use to perform these DNA edits and why?
VEDS, is associated with pathogenic mutations in the COL3A1 gene, arising as disruptions with the glycine residues needed for the correct formation of collagen. You could use CRISPR CAS9, to design an RNA capable to target the mutated region and introduce a donor repair template with the correct sequence. Alternatively, base editing could be use to change the erroneous base.
Week 3 HW: Lab Automation
I designed a drawing with the website that was made available to us https://opentrons-art.rcdonovan.com/. It made it easier to get used to the way the points would be done inside the plate to create our art. Personally I decided to create a flower, took the exact points for each color, green (sfGFP) and red (mrFP1). I integrated the points to the base code, and customized it for the design to be possible, especially with the high quantity of points and the limit of the 20ul pipette.
1. Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.
In this paper, they use Opentrons to automate the assembly of bisoensors. The team created a flouride responsive genetic biosensor that produces a measurable florescent signal when positive. They compared ht precision and consistency of hte building between manually prepare and the automatic assembly done by the robot, which ended up giving as realible results. The paper highlights how the automatization can increase throughtput, reduce human error, and standardize the workflows in synthetic biology for the development and screening of biosensors.
2. Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode, Python scripts, 3D printed holders, a plan for how to use Ginkgo Nebula, and more. You may reference this week’s recitation slide deck for lab automation details.
For my final project ideas, Im thinking of creating biosensors for multiple things, so essentially I could use as an example the paper, and create a code to work with Opentrons to create the workflow of the engineering and producing them. To create the biosensor for biomarkers, and being a comitted listener distnat from the node, automatization robots are my chance to do the wet lab of the synthetizing of the assembling of the circuit for biosensor, quantify the flourensence, and create data dirven profiling.
Week 4 HW: Protein Design Part 1
PART A: CONCEPTUAL QUESTIONS
1.How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)
2.Why do humans eat beef but do not become a cow, eat fish but do not become fish?
3.Why are there only 20 natural amino acids?
4.Can you make other non-natural amino acids? Design some new amino acids.
5.Where did amino acids come from before enzymes that make them, and before life started?
6.If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?
7.Can you discover additional helices in proteins?
8.Why are most molecular helices right-handed?
9.Why do β-sheets tend to aggregate?
-What is the driving force for β-sheet aggregation?
10.Why do many amyloid diseases form β-sheets?
-Can you use amyloid β-sheets as materials?
11.Design a β-sheet motif that forms a well-ordered structure.
