First, describe a biological engineering application or tool you want to develop and why
Pattern-Based Rapid Diagnostic Platform for Dengue Virus: A rapid diagnostic platform for dengue virus (DENV) that integrates innate immune recognition, molecular recognition, and biosensor engineering to address key limitations of existing diagnostic methods. The proposed system combines mannose-binding lectin for the recognition of viral glycoproteins, dengue-specific aptamers targeting conserved regions of viral proteins, and signal transduction through a portable biosensor to enable rapid readout. This approach is motivated by the fact that current dengue diagnostics are often expensive and exhibit reduced sensitivity and reliability in dengue-endemic regions, particularly in countries like mine (Colombia), where prior flavivirus exposure compromises serological test performance and access to reliable diagnostics is limited by public healthcare infrastructure (Terenteva et al., 2025).
Opentrons Artwork opentrons-art.rcdonovan.com/?id=oevp91e27i3m061
Post-Lab Questions Find and describe a published paper that utilizes the Opentrons This article combines an open‑source liquid‑handling robot (Opentrons OT‑One‑S Hood) with four interchangeable modules that perform magnetic‑bead DNA isolation, isothermal recombinase polymerase amplification (RPA) of the ctrA gene, exonuclease digestion to generate single‑stranded DNA, and detection on a paper‑based vertical‑flow microarray (VFM) using anti‑biotin gold nanoparticles for colorimetric read‑out.
Subsections of Homework
Week 1 HW: Principles and Practices
First, describe a biological engineering application or tool you want to develop and why
Pattern-Based Rapid Diagnostic Platform for Dengue Virus: A rapid diagnostic platform for dengue virus (DENV) that integrates innate immune recognition, molecular recognition, and biosensor engineering to address key limitations of existing diagnostic methods. The proposed system combines mannose-binding lectin for the recognition of viral glycoproteins, dengue-specific aptamers targeting conserved regions of viral proteins, and signal transduction through a portable biosensor to enable rapid readout. This approach is motivated by the fact that current dengue diagnostics are often expensive and exhibit reduced sensitivity and reliability in dengue-endemic regions, particularly in countries like mine (Colombia), where prior flavivirus exposure compromises serological test performance and access to reliable diagnostics is limited by public healthcare infrastructure (Terenteva et al., 2025).
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.
GOAL 1:
Ensure that diagnostic accuracy supports appropriate clinical decision-making, minimizing the risk of misdiagnosis, delayed care, and public health mismanagement.
Subgoals:
Diagnostic Reliability: Standards validated in endemic populations and ongoing monitoring of false positives and false negatives.
Prevention of clinical misinterpretation: Support test results with clear and accessible interpretive guidance.
GOAL 2:
Ensure to promote equitable access and global health justice in the development and use of the diagnostic technology.
Subgoals:
Affordability and Accessibility: Promote public–private collaboration for the deployment of dengue diagnostics in high-burden countries, without dependence on specialized infrastructure.
Prevent Technological Exclusion: Ensure that the diagnostic tool is usable in decentralized healthcare settings, such as rural clinics and community health centers
3.Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”)
Establish context-specific validation requirements for diagnostic deployment:
The objective of this action is to prevent clinical harm caused by diagnostic failures that persist under current practices in endemic settings, particularly false negatives in secondary dengue infections and false positives due to flavivirus cross-reactivity.
To achieve this, regulatory agencies and public health institutions should require that rapid dengue diagnostics be validated directly in endemic communities—especially among people with prior flavivirus exposure—before they’re approved or rolled out. This means shifting approval pathways so they rely on real‑world performance data, not just controlled lab studies or trials in non‑endemic settings.
Assuming that regulators can review performance data in the specific contexts where diagnostics will be used, that accuracy can vary across different populations, and that manufacturers will adapt their designs to meet these requirements. Even so, a “false success” could occur if compliance is limited to minimal testing in some endemic populations. True success would mean diagnostics that work reliably across diverse populations, helping to reduce misdiagnosis and support appropriate clinical decisions.
Ensuring Transparency and Open Validation in Diagnostic Development:
The purpose of this action is to promote responsible innovation and build trust by ensuring that the limitations of diagnostics are openly documented and shared before large-scale deployment. By making failure modes, cross-reactivity profiles, and other constraints visible early, developers, regulators, and clinicians can make better-informed decisions and reduce risks to patients and public health.
Funding agencies and scientific journals should require transparent reporting of assay limitations as part of the evaluation and publication process. Shared validation datasets could be established. Incentives—such as eligibility for specific funding programs, prioritized review, or formal recognition—can encourage participation from researchers and companies while improving the overall reliability and comparability of diagnostic technologies.
This approach assumes that increased transparency improves diagnostic quality and that researchers and companies will share performance data to reduce failure across the sector. Potential failure points include limited industry participation due to intellectual property concerns or competitive pressures. Success is defined by establishing routine, independent testing of diagnostic performance claims, creating a standard expectation of reliability prior to clinical adoption.
4.Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals. The following is one framework but feel free to make your own:
–>
Does the option:
Option 1
Option 2
Option 3
Enhance Biosecurity
• By preventing incidents
X
• By helping respond
X
Foster Lab Safety
• By preventing incident
X
• By helping respond
X
Protect the environment
• By preventing incidents
X
• By helping respond
X
Other considerations
• Minimizing costs and burdens to stakeholders
X
• Feasibility?
X
• Not impede research
X
• Promote constructive applications
X
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
Based on the above, the following actions would be prioritized:
Establish context-specific validation requirements for dengue diagnostics.
This action has the highest priority because it directly ensures diagnostic reliability in endemic populations. Requiring validation in communities with prior flavivirus exposure reduces false negatives, false positives, and clinical mismanagement. It provides the regulatory foundation necessary for safe, equitable deployment. Without this step, large-scale implementation could compromise patient care and public health decision-making.
Ensure transparency and open reporting of diagnostic limitations.
This action strengthens accountability and trust by requiring disclosure of performance data, cross-reactivity profiles, and failure modes. However, its effectiveness depends on clear validation standards. Properly designed, it improves long-term reliability and supports informed clinical use, while balancing industry participation and innovation incentives.
Ethical concerns that arose, especially any that were new to you.
This week, I learned that developing a diagnostic platform is more than just a technical or experimental challenge; it is also a matter of governance and biosecurity—areas that were largely new to me. Previously, I focused mainly on protocol design, molecular mechanisms, and performance metrics. In class and doing the homework, I began to understand that every diagnostic tool exists within a broader regulatory, ethical, and public health framework that determines how it is validated, deployed, and monitored in real-world settings.
One key ethical concern that emerged for me is that diagnostic errors are not just laboratory inaccuracies—they can produce systemic harm. Poor validation, lack of transparency, or weak oversight can lead to misdiagnosis, inequitable access, and loss of public trust.
To address these issues, some governance actions that I think should be required are context‑specific validation before any large‑scale deployment, establishing clear transparency standards to ensure diagnostic limitations are openly reported, and implementing post‑market monitoring to quickly identify and respond to performance gaps.
References
Terenteva, S., Golani-Zaidie, L., Avivi, S., Lustig, Y., Indenbaum, V., Koren, R., Hoa, T. M., Tuyen, T. T. K., Huyen, M. T., Hoan, N. M., Hoi, L. T., Trung, N. V., Schwartz, E., & Danielli, A. (2025). Sensitivity and Cross-Reactivity analysis of Serotype-Specific Anti-NS1 serological assays for dengue virus using optical modulation biosensing. Biosensors, 15(7), 453. https://doi.org/10.3390/bios15070453
WEEK 2 LECTURE PREP
Questions from Professor Jacobson
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 replicative DNA polymerases is roughly 10⁻⁵ errors per nucleotide incorporated. With proofreading (3’→5’ exonuclease activity), this improves to about 10⁻⁷, and after post-replicative mismatch repair, the final error rate drops to approximately 10⁻⁹ to 10⁻¹⁰ per base per replication cycle.
Comparing that to the human genome, which is about 3 × 10⁹ base pairs per haploid. If replication occurred at 10⁻⁵ error frequency with no correction, that would mean tens of thousands of mutations per cell division, incompatible with genomic stability. Even at 10⁻⁷, you would expect hundreds of mutations per division.
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?
An average 400 amino acids long. Because of the degeneration of the genetic code, most amino acids are specified by multiple synonymous codons.
In practice, most of these sequences do not function equivalently because:
1. Codon usage bias
2. mRNA secondary structure
3. GC content constraints
4. Regulatory elements within coding regions
Questions from Dr. LeProust
What’s the most commonly used method for oligo synthesis currently?
The most common method for oligo synthesis is solid-phase phosphoramidite chemistry. Nucleotides are added stepwise in the 3’→5’ direction on a solid support, with cyclic coupling, capping, oxidation, and deprotection steps.
Why is it difficult to make oligos longer than 200nt via direct synthesis?
Because each coupling step is not 100% efficient. The yield drops with length. At 99% efficiency per step, a 200-mer has only a few full-length products. Beyond that, truncated products dominate, and purification becomes inefficient.
Why can’t you make a 2000bp gene via direct oligo synthesis?
Because the cumulative stepwise loss would make the full-length product essentially nonexistent. Long genes are built by assembling shorter oligos not by single continuous chemical synthesis.
Question from George Church
What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?
The 10 essential amino acids in animals are: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, and arginine. These cannot be synthesized de novo and must be obtained from the diet.
Respect “Lysine Contingency”:
Lysine is not the only essential amino acid; it is one of several indispensable amino acids. This means that engineering a specific dependence on lysine is conceptually no different from creating dependence on any other essential amino acid. Its practical value does not lie in biochemical uniqueness, but in its controllability: environmental availability can be tightly regulated, making it a useful strategy.
Source: ChatGPT (Open AI)
Because several codons encode the same amino acid, but the frequency at which these codons are used varies among organisms. Each species has preferences for particular codons, a phenomenon known as codon usage bias.
When expressing a gene from one organism in a different host without prior codon optimization, translation efficiency can be reduced, leading to lower protein production or even affecting proper protein folding.
Which organism have I chosen to optimize the codon sequence for, and why?
I chose to optimize the sequence for Escherichia coli because it is one of the most widely used systems for recombinant protein production. It is easy to cultivate, grows quickly, and is cost‑effective. In addition, there are well‑established genetic tools that enable efficient protein expression when codons are adapted to its natural codon bias, and it is the organism I am most familiar with.
You have a sequence! Now what?:
What technologies could be used to produce this protein from your DNA?
Recombinant gene expression in bacteria, yeasts, or cell‑free systems
Cell-dependent transcription and translation
The gene is inserted into a plasmid and then introduced into a host organism.
The process begins with cloning, where the codon‑optimized gene is inserted into a plasmid. During transformation or transfection, the plasmid is delivered into the host cell. Inside the cell, the host RNA polymerase recognizes the promoter and transcribes the DNA into mRNA. This mRNA is then read by ribosomes, which synthesize the protein by assembling amino acids according to the codon sequence. Finally, the newly synthesized protein folds and, if necessary, undergoes further modifications.
MBL for be used as an initial viral capture molecule due to its affinity for high-mannose glycans present on the viral envelope protein.
What technology or technologies would you use to perform sequencing on your DNA and why?
To sequence DNA, I would use Illumina sequencing for its high accuracy and cost-effectiveness, since my idea is targeted gene analysis and Illumina technology provides low error rates and high throughput, making it ideal for detecting deletions.
Is your method first-, second or third-generation or other? How so?
Second generation, because it performs massive sequencing in parallel, use clonal amplification (bridge amplification) before reading and is necessary perform the amplification before sequencing
What is your input? How do you prepare your input? List the essential steps.
Input: MBL2
Preparation: Using linear synthetic DNA MBL2 sequence
End repair.
A-tailing.
Adapter ligation for Illumina-compatible adapters
PCR enrichment of adapter-ligated fragments.
Cleanup and quantification.
What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample?
Essential steps:
Flow Cell Binding
MBL2 gene fragments with adapters are loaded onto the flow cell.
The fragments hybridize to complementary oligonucleotides immobilized on the surface.
Bridge Amplification
Each fragment is locally amplified, forming clonal clusters, generating many identical copies of each fragment, increasing the detectable signal during sequencing.
Sequencing by Synthesis
Fluorescently labeled nucleotides with reversible terminators.
In each cycle:
• A labeled nucleotide (A, T, C, or G) is added.
• Only one nucleotide is incorporated per cycle.
• A camera detects the emitted fluorescence.
• The system records the corresponding color.
• The fluorophore and chemical blocking group are then removed.
• The cycle is repeated.
Base calling performed through:
Optical detection of the fluorescent signal in each cluster.
Each color corresponds to a specific base (A, T, C, or G), the software converts the light signal into a nucleotide sequence, and a quality score is assigned to each base.
What is the output of your chosen sequencing technology?
Millions of short reads. Generated in FASTQ format files, with the nucleotide sequence of each read and a quality score assigned to every base.
WRITE
What DNA would you want to synthesize (e.g., write) and why?
would synthesize a codon-optimized version of the MBL2 coding sequence (CDS) to produce recombinant MBL protein for use as a viral capture molecule in a diagnostic assay.
What technology or technologies would you use to perform this DNA synthesis and why?
Array-based or column-based phosphoramidite DNA synthesis. Because it is a method with high sequence fidelity and control over sequence design
What are the essential steps of your chosen sequencing methods?
a) Solid-phase attachment
b) Deprotection
c) Coupling
d) Capping
e) Oxidation
f) Cleavage and deprotection
g) Sequence verification
What are the limitations of your sequencing method in terms of speed, accuracy, scalability?
In terms of accuracy, each coupling step is not 100% efficient, so errors can accumulate as the sequence length increases. Regarding length, synthesis is limited to about 150–200 base pairs per oligonucleotide, meaning longer genes must be assembled from multiple overlapping fragments. In terms of speed, synthesis is relatively fast for short oligos, but full gene synthesis requires extra assembly and validation steps,increasing turnaround time. Concerning scalability, array-the individual yield per oligo may be lower compared to column-based synthesis. Finally, cost increases with more complex constructs due to the need for assembly and error correction.
EDIT
What DNA would you want to edit and why?
MBL2 gene, which encodes Mannose-binding lectin, to enhance its expression in individuals with naturally low serum MBL levels. Increased MBL expression could potentially improve early immune recognition of viral pathogens
What kinds of edits might you want to make to DNA? Why?
Precise regulatory modification rather than altering the protein-coding sequence. Introducing promoter variants associated with higher transcriptional activity could increase protein levels without changing protein structure, minimizing unintended functional consequences while enhancing host defense.
What technology or technologies would you use to perform these DNA edits and why?
CRISPR-Cas9 with Homology-Directed Repair, because the precise control over the type of genetic modification and flexibility depending on whether the goal is a single-base change or a larger regulatory insertion.
How does your technology of choice edit DNA? What are the essential steps?
Using a guide RNA (gRNA) to direct the Cas9 nuclease to a specific genomic sequence.
Target recognition
DNA cleavage
DNA repair
What preparation do you need to do and what is the input for the editing?
Design a specific guide RNA targeting for MBL2, verifying minimal off-target sites using bioinformatic tools.
Inputs: Cas9 protein, Guide RNA, delivery system and target cell
What are the limitations of your editing methods (if any) in terms of efficiency or precision?
Efficiency:
HDR efficiency is often low in non-dividing cells, and editing rates depend on the cell type and genomic context.
Precision:
Potential off-target edits if the guide RNA is not highly specific and NHEJ repair may introduce unintended insertions or deletions.
Week 2 HW: Lab Automation
Opentrons Artwork
opentrons-art.rcdonovan.com/?id=oevp91e27i3m061
Post-Lab Questions
Find and describe a published paper that utilizes the Opentrons
This article combines an open‑source liquid‑handling robot (Opentrons OT‑One‑S Hood) with four interchangeable modules that perform magnetic‑bead DNA isolation, isothermal recombinase polymerase amplification (RPA) of the ctrA gene, exonuclease digestion to generate single‑stranded DNA, and detection on a paper‑based vertical‑flow microarray (VFM) using anti‑biotin gold nanoparticles for colorimetric read‑out.
Magnetic Dynabeads capture pathogen DNA from cerebrospinal fluid or water samples, after which the beads are washed and the DNA is released into the amplification mix 3. The RPA reaction runs at 37 °C, using a biotin‑labelled forward primer and a 5′‑phosphate reverse primer; the resulting double‑stranded amplicons are digested by Lambda exonuclease, which removes the phosphorylated strand and leaves a biotin‑tagged single strand for hybridisation. The single‑stranded amplicons are applied to nitrocellulose VFM spots that contain capture probes for ctrA; anti‑biotin gold nanoparticles bind the biotin tag, and a signal‑enhancement solution produces a visible colour change at positive spots.
ecause all steps are scripted on the Opentrons platform, the robot performs liquid handling automatically, reducing hands‑on time to 110 min for eight samples—about 18 % faster than manual processing—and lowering consumable cost to roughly USD 16 per sample. The open‑source hardware and standard lab consumables make the system adaptable to other pathogens and suitable for deployment in low‑resource settings.
Write a description about what you intend to do with automation tools for your final project
For my final project, I intend to develop a low-cost diagnostic test for the detection of Dengue virus (DENV), optimized for use in low-resource settings. The system would integrate:
A lateral-flow or paper-based detection platform
Automation in the creation and production of the test
The goal is to reduce human error, increase reproducibility, and lower production costs while maintaining diagnostic sensitivity and specificity.
