JiaheSun — HTGAA Spring 2026
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Contact info
Homework
- Week 1 HW: Principles and Practices
- Week 2 HW: DNA Read, Write, and Edit
- Week 3 HW: Lab Automation
- Week 4 HW: Principles and Practices
Subsections of JiaheSun — HTGAA Spring 2026
Homework
Weekly homework submissions:
Week 1 HW: Principles and Practices
- Introduction With a rather limited background in the field of synthetic biology and bioengineering, I sketched out my initial scope of interest in closed-loop controllers, in which they are autonomous and adjust to the environment around. While I’m also interested in the bidirectional communication via the gut-brain axis. I want to explore the idea of engineering a gut bacterium with a synthetic genetic circuit that could detect biomarkers in the gut and conditionally produce neuroactive compounds that modulate brain activity via the GBA.
Week 2 HW: DNA Read, Write, and Edit
3.1. Choose your protein. In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, google), obtain the protein sequence for the protein you chose. I have selected PIEZO1 as my protein, that is a protein sitting in the cell membrane and opens when the membrane is physically stretched, compressed, or deformed, basically detecting the membrane tension.
Post Lab Questions Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode or Python scripts, procedures you may need to automate, 3D printed holders you may need, and more. Example ideas that you can create a protocol for: Use the cloud laboratory to screen an array of biosensors constructs that you design, synthesize, and express using cell-free protein synthesis Use Opentrons to dispense microorganisms onto fabric to design “living textiles” as “bio artwork”
Week 4 HW: Principles and Practices
Part A. Conceptual Questions 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) Why do humans eat beef but do not become a cow, eat fish but do not become fish? Why are there only 20 natural amino acids? Can you make other non-natural amino acids? Design some new amino acids. Where did amino acids come from before enzymes that make them, and before life started? If you make an α-helix using D-amino acids, what handedness (right or left) would you expect? Can you discover additional helices in proteins? Why are most molecular helices right-handed? Why do β-sheets tend to aggregate? What is the driving force for β-sheet aggregation? Why do many amyloid diseases form β-sheets? Can you use amyloid β-sheets as materials? Design a β-sheet motif that forms a well-ordered structure.
Subsections of Homework
Week 1 HW: Principles and Practices
1. Introduction
With a rather limited background in the field of synthetic biology and bioengineering, I sketched out my initial scope of interest in closed-loop controllers, in which they are autonomous and adjust to the environment around.
While I’m also interested in the bidirectional communication via the gut-brain axis. I want to explore the idea of engineering a gut bacterium with a synthetic genetic circuit that could detect biomarkers in the gut and conditionally produce neuroactive compounds that modulate brain activity via the GBA.
The circuit should ideally consist of a sensor module, processing module, and a response module. The logic is elucidated as following:
Inflammation detected → threshold exceeded → produce calming molecules → inflammation decreases → production shuts off.
This idea draws distinction from those open-loop, stress-relieving gummies and pills in that, this is a self-regulating therapeutic that produces compounds at the site where the gut-brain signaling infrastructure exists, and only produces upon conditional activation when the stress/inflammation biomarker exceeds a certain threshold.
2. Governance Goals
The overarching goal is Non-Malfeasance (preventing harm)
The nature of the technology involves releasing a genetically engineered organism into the human body, and potentially into the broader environment, making harm prevention and the Dual Use Research Concern (DUrC) indispensable presences and should be carried out at multiple scales.
SubGoal 1A: Preventing Uncontrolled Spread and Ecological Contamination
The engineered microbe must not exist beyond its therapeutic window, which means it should by no means spread to unintended hosts, or transfer its synthetic genes to wild microbial populations via the following possible routes:
- Horizontal gene transfer (HGT): Synthetic circuit components (especially antibiotic resistance markers used in cloning) could transfer to pathogenic gut bacteria.
- Environmental shedding: Engineered bacteria will be excreted and enter wastewater and soil ecosystems.
- Mutation: The organism could evolve and mutate overtime to the point where the original means of control no longer works, or it can gain unintended functions.
SubGoal 1B: Preventing Negative Neurological/Immunological Effects
The closed-loop circuit must not overproduce compounds that trigger immune reactions within the body or interferes with the existing microbiome in unintended ways, such as:
- Overproduction toxicity: A sensor that is too sensitive or a failed threshold filter could flood the gut with GABA/serotonin precursors.
- Immune overactivation: The engineered organism might trigger inflammatory responses, paradoxically worsening the target condition.
- Microbiome disruption: The engineered organism at therapeutic densities could outcompete native beneficial bacteria.
SubGoal 1C: Informed Consent
Governance must address who gets access and whether patients can meaningfully consent to hosting a living engineered organism, as the commitment is larger than taking in a single pill.
3. Potential Actions
Three potential governance actions are considered below, incorporating 1) Purpose, 2) Design, 3) Assumptions, and 4) Risk of Failure and “Success”.
Governance Action 1: Comprehensive policy framework and clear assignment on roles played by different actors
Purpose: The work conducted with living organisms in making them biotherapeutic product usually fall under FDA’s established framework of CBER, but due to the closed-loop nature of the synthetic circuit, there are no detailed requirements/regulations revolving around how to exert controllable influence that distinguishes from the treatment of those open-looped projects.
Design: Given the participation of various actors, when FDA issues the guidance, academic labs should design/provide corresponding biocontainment tools. While biotech companies comply and absorb testing costs. Research agencies should then standardize biocontainment toolkits to lower barriers for smaller labs. Cross-agency coordination with environmental protection agencies (e.g. EPA) may be needed.
Assumptions
- Effective switches can be engineered over time to keep the microbiome in check
- FDA has sufficient synbio experts in evaluating the circuit design
- In vitro stability testing predicts in vivo behavior
Risks
- Failure: IF the standards were set too high making the project difficult to perform, it could lead to the decline in industry as small labs and startups may choose to opt out.
- Success: A standard designed too well could lead to underestimation of risks.
Governance Action 2: Long Term Monitoring and Clinical Trials
Purpose: Given the closed-loop nature and the potential changes that could occur in living therapeutics, clincal trial framework should establish different tiers that occurs over a designated timescale for constant surveillance.
Design: The clinical trials should develop at least three tiers, with
- Tier 1 (1-3 yr): Standard testing phase
- Tier 2 (5 yr): Mandatory microbiome monitoring and tracking of genomic sequences
- Tier 3: Constant survillance of wastewater disposal in experimenting/trial regions
Assumptions
- Patient will remain in 5 year follow up
- The engineered organism can be effectively tracked within gut environment
Risks
- Failure: Unforseen development of organism is sighted after widespread distribution.
- Success: Over institutionalized framework could slow development of future iterations.
Governance Action 3: Transparency and International Oversee
Purpose: In considering the potential widespread use of such ideation, the public should gain transparency to the fundamental logic/codes. Simultaneously, international harmonization groups like WHO should develop and align the set of harmonized minimum standards for testing and monitoring.
Design: National governments in coordinating and aligning regulations under international organizations and synbio industry leaders. Commited collaboration between public and private sectors in a foreseeable timescale.
Assumptions
- Committed support among decision maker exists despite current issue in international relations.
- Applicable universal standard despite different cultural practice
- Development of technology be in pace with international harmonization.
Risks
- Failure: No actual efforts of enforcement made.
- Success: Rigorous standards that further stabilize the advantage of developed countries, and enlarge the medical development and accessibilities between countries.
4. Scoring Framework
The following rubric evaluates the governance options presented above on a 1–3 scale (1=week/limited, 2=moderate, 3=strong) across the span of biosecurity, lab safety, environmental protection, and practical considerations.
| Does the option: | Option 1 | Option 2 | Option 3 |
|---|---|---|---|
| Enhance Biosecurity | |||
| • By preventing incidents | 3 | 2 | 2 |
| • By helping respond | 1 | 3 | 3 |
| Foster Lab Safety | |||
| • By preventing incident | 3 | 2 | 2 |
| • By helping respond | 2 | 2 | 1 |
| Protect the environment | |||
| • By preventing incidents | 3 | 2 | 2 |
| • By helping respond | 1 | 3 | 3 |
| Other considerations | |||
| • Minimizing costs and burdens to stakeholders | 2 | 2 | 1 |
| • Feasibility? | 2 | 3 | 1 |
| • Not impede research | 1 | 2 | 2 |
| • Promote constructive applications | 2 | 3 | 3 |
| Total | 20 | 24 | 20 |
5. Prioritized Option
Given the overall scoring, Governance Action 2 yields the highest total amongst the three, because the design in stages of trial over a timescale monitors the progress of experiment closely and allows for early detection of incidents. The gradual development also allows brings the market into consideration, making the idea of wide application possible.
However, it also contain weakness that needs to be accompanied by complementary actions. Specifically on prevention, Action 1 scores higher in that it implants kill switches in the initial engineering phase.
Action 3 touches a little bit of everything, but it should be of a later consideration when the technology and domestic standards became more mature, as implementing regulations on an international level generates huge costs and often require longer time for reconciliation/negotiation.
Assignment:
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, according to slide 8, is 1:10^6. The human genome as noted is 3.2 billion base pairs (gbp), and hence if we were to do the calculation there would be around three thousand new mutations/cell division. The biology deals with the discrepancy through error correction like MutS Repair System, that detects the mismatched base pairs and resynthesize it correctly, therefore bringing down the error rate and enabling the copying to proceed with very few/zero errors.
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 human protein is encoded by around 1036 base pairs of DNA (slide 6), and divided by three (codon) will get roughly around 345 amino acids/protein. So given the number, there’s around 10^150 possible DNA sequences that result in the same primary chain of amino acids. But the majority are redundant, and in some situations a sequence of amino acid would create mRNA structures like hairpin that blocks the ribosome from binding and the forming of right protein.
Questions from Professor LeProust
What’s the most commonly used method for oligo synthesis currently?
The most used method is the phosphoramidite method, which is a 4 step chemical cycle that repeats for N times, specifically including coupling (with phosphoramidite), capping (unreacted sites), oxidation, and deblocking.
Why is it difficult to make oligos longer than 200nt via direct synthesis?
It is difficult mainly due to the inefficiency of the coupling steps and the accumulation of errors, given the exponentially decaying yield, as the error rate accumlates, the majority would be of failure sequence by the time it reaches 200.
Why can’t you make a 2000bp gene via direct oligo synthesis?
Because the direct oligo synthesis is performed via phosphoramidite, and due to the multiplicative nature of the success rate and the final yield follows an exponential decay curve, as the number of nucleotides increases, the accuracy will go down. By the time it reaches 2000, it would be hardly possible to extract the correct sequence among all disturbances and noises. Hence bioengineers synthesize smaller oligos and stitch them together to ensure the correct sequence.
Question from Professor 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 acid (from the slide and with the aid of google) are listed below:
- Arginine (Arg)
- Histidine (His)
- Isoleucine (Ile)
- Leucine (Leu)
- Lysine (Lys)
- Methionine (Met)
- Phenylalanine (Phe)
- Threonine (Thr)
- Tryptophan (Trp)
- Valine (Val)
The Lysine Contingency (according to Google) refers to the genetic alteration performed in the movie Jurassic Park, that made dinosaurs unable to produce lysine, therefore relying on human supplements to survive. But this idea does not stand as it is an essential amino acid within them that doesn’t need to be synthesized, and hence dinosaurs can gain lysine by eating other organisms. This idea sheds light on the biocontainment method of NSAA (non standard amino acid), which organisms cannot obtain in a natural setting, and hence is a more secure contingency.
Week 2 HW: DNA Read, Write, and Edit
3.1. Choose your protein.
In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, google), obtain the protein sequence for the protein you chose.
I have selected PIEZO1 as my protein, that is a protein sitting in the cell membrane and opens when the membrane is physically stretched, compressed, or deformed, basically detecting the membrane tension.
Protein Sequence (2,521 aa)
Click to expand full protein sequence
Key features: 38 transmembrane helices/monomer · Trimeric propeller architecture · ~900 kDa functional complex
3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.
The Central Dogma discussed in class and recitation describes the process in which DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.
Native DNA Sequence (7,566 bp)
Click to expand native DNA coding sequence
3.3. Codon optimization.
Once a nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize google for a “codon optimization tool”. 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?
- E. coli Codon-Optimized DNA (7,566 bp)
Optimized for expression in E. coli C43(DE3). Rare codons (AGG/AGA for Arg, CUA for Leu, AUA for Ile) replaced with E. coli-preferred synonymous codons to prevent ribosomal stalling and improve yield.
Click to expand E. coli-optimized sequence (codon-spaced)
Key differences from human-optimized version: Arginine codons AGG/AGA → CGT/CGC (abundant E. coli tRNAs) · Leucine CTA → CTG/CTT · Isoleucine ATA → ATT · Lower GC content (~52% vs ~69% in human-optimized)
Quick Comparison
| Property | Protein | Native DNA | E. coli-Optimized DNA |
|---|---|---|---|
| Length | 2,521 aa | 7,566 bp | 7,566 bp |
| GC content | — | ~58% | ~52% |
| Target host | — | H. sapiens | E. coli C43(DE3) |
| Rare codons | — | None (native) | Eliminated |
| Encoded protein | PIEZO1 | Identical | Identical |
Note: Both DNA sequences encode the exact same protein. Only the synonymous codon choices differ, optimized for the translational machinery of the target host organism.
Week 3 HW: Lab Automation
Post Lab Questions
Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode or Python scripts, procedures you may need to automate, 3D printed holders you may need, and more.
Example ideas that you can create a protocol for: Use the cloud laboratory to screen an array of biosensors constructs that you design, synthesize, and express using cell-free protein synthesis Use Opentrons to dispense microorganisms onto fabric to design “living textiles” as “bio artwork”
Find and briefly summarize a published paper that utilizes laboratory automation to achieve novel biological applications Include in your summary: General overview (2 paragraphs) Findings (1 paragraph) Relevant Figures (1 - 2 max)
Week 4 HW: Principles and Practices
Part A. Conceptual Questions
- 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)
- Why do humans eat beef but do not become a cow, eat fish but do not become fish?
- Why are there only 20 natural amino acids?
- Can you make other non-natural amino acids? Design some new amino acids.
- Where did amino acids come from before enzymes that make them, and before life started?
- If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?
- Can you discover additional helices in proteins?
- Why are most molecular helices right-handed?
- Why do β-sheets tend to aggregate?
- What is the driving force for β-sheet aggregation?
- Why do many amyloid diseases form β-sheets?
- Can you use amyloid β-sheets as materials?
- Design a β-sheet motif that forms a well-ordered structure.
Labs
Lab writeups:
Image 1 (Mid-run photograph): The photograph taken during electrophoresis shows the gel submerged in TAE within the gel box. Two colored dye fronts are faintly visible — a blue band and a dark purple band — but they appear localized to only one or two lanes. The majority of the gel appears empty, with no visible dye migration in the other wells. This is already an early indicator that most wells were either not loaded successfully or contained insufficient DNA.
Subsections of Labs
Week 1 Lab: Pipetting
Week 2 Lab: DNA Gel Art
Image 1 (Mid-run photograph): The photograph taken during electrophoresis shows the gel submerged in TAE within the gel box. Two colored dye fronts are faintly visible — a blue band and a dark purple band — but they appear localized to only one or two lanes. The majority of the gel appears empty, with no visible dye migration in the other wells. This is already an early indicator that most wells were either not loaded successfully or contained insufficient DNA.
Image 2 (GeneSnap image): The final imaging result is largely dark. Only a single lane shows any detectable fluorescence — a faint, somewhat smeared signal concentrated in what appears to be one lane, with no clearly resolved discrete bands. The remaining lanes are entirely blank. This represents an unsuccessful gel run in terms of the intended gel art pattern.
Analysis of What Went Wrong Based on the observations made during lab sessions and the photographic evidence, several compounding factors likely contributed to the result:
- Pipetting error during well loading. When I was loading the fourth slot, the pipette tip was not properly inserted into the well. This is a critical failure point. In submerged gel electrophoresis, the wells are filled with buffer. The loading dye’s density causes the sample to sink — but only if it is dispensed directly into the well. If the tip hovers above the well or is positioned outside it, the sample disperses into the surrounding buffer and is effectively lost. This likely explains why most lanes are empty on the final image.
- Insufficient electrophoresis run time due to electrical issues. There was an unforeseen electrical short circuit that cut the run time short. This is consistent with the imaging result — even in the one lane that has signal, the DNA has not migrated very far, and there is no clear band resolution. A truncated run means fragments have not separated sufficiently, resulting in a compressed, smeared appearance rather than discrete bands. The faint dye fronts visible in Image 1 also suggest limited migration distance.
- Potential variability in reaction preparation. Another plausible explanation adding to the result could be the differences in mixing or component proportions across the PCR tubes. This is plausible as if the Lambda DNA stock was not thoroughly vortexed or flicked, concentration could vary between tubes. Similarly, enzyme or buffer pipetting errors at the 1–3 μL scale are common and can result in incomplete digestion or no digestion at all, though the imaging suggests the bigger problem was DNA not being present in the wells at all.
- Low overall signal intensity. Even the one visible lane is quite faint. This could indicate that the total DNA mass loaded was below the detection threshold of SYBR Safe under blue light excitation. With 1.5 μg of Lambda DNA per reaction and SYBR Safe staining, bands should normally be clearly visible. The faintness suggests either DNA was lost during loading, the stain was not adequately mixed into the gel, or the transilluminator exposure settings were suboptimal.