Week 1 HW: Principles and Practices

1. First, describe a biological engineering application or tool you want to develop and why.

I want to develop a highly efficient bacterial chassis for rapid intracellular biosynthesis of novel PHA (polyhydroxyalkanoate) copolymers. More than 150 different hydroxyalkanoate monomers have been identified, and they can be combined into co-polymers (and potentially ter-/quad-polymers) with variable composition and sequence/microstructure, leading to an astronomical design space.

My goal is to build a chassis that makes the design–build–test loop fast and reliable: (1) use computational/AI approaches and literature review to prioritize promising copolymer compositions for target properties, then (2) rapidly prototype biosynthesis in a standardized host with predictable performance, and (3) generate experimental data to validate predicted properties and improve models.

This tool idea is directly inspired by my work: I’m the CEO of Bioplastix, a biotech startup with the mission to accelerate the transition to biodegradable bioplastics. We already have a promissing co-polymer (PLA-PHB with different proportions of PLA) and a highly efficient E.coli chassis to produce it. We want to accelerate the discovery of new co-polymers. A chassis that can quickly produce and screen new PHA copolymers would let us create radically better biopolymers and accelerate transition to bioplastics.

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.

The goal of this biological tool is to contribute to an ethical future by reducing harm to human and planetary health through the replacement of oil-based plastics. Conventional plastics accumulate in ecosystems and human bodies (Nihart, A.J., Garcia, M.A., El Hayek, E. et al. Bioaccumulation of microplastics in decedent human brains. Nat Med 31, 1114–1119 (2025)), have high associated carbon footprints, and often rely on toxic ingredients across their life cycle.

For this tool to be successful, I suggest two core policy and governance goals:

Goal 1: Encourage the adoption of bio-based and biodegradable plastics. At present, biodegradable bioplastics such as PHAs remain significantly more expensive than oil-based plastics (e.g., PHAs at approximately 4–5 USD/kg versus PET at ~1.3 USD/kg). Policies that reduce this gap during early scale-up phases are therefore essential to enable meaningful market penetration.

Goal 2: Prevent harm from new biological strains and novel polymer compositions. Because this platform enables the rapid creation of many new copolymers and engineered microbial strains, governance is critical to avoid unintended biological or environmental consequences. Like any powerful biological technology, it must operate within clearly defined biosafety, material safety, and ethical boundaries.

Note: I could state Goal 1 as: Ban traditional plastics or sidcourage the use of traditinal plastics. A global ban or restrictions on conventional plastics could, in principle, accelerate this transition, but such an outcome appears unlikely in the near term, as illustrated by the limited progress of the UN Global Plastics Treaty negotiations.

3. Next, describe at least three different potential governance “actions” by considering four aspects (Purpose, Design, Assumptions, Risks of Failure & “Success”).

Action 1: Tax incentives for biodegradable and bio-based plastics

Purpose: Today, in most countries and subnational jurisdictions, there are no meaningful tax incentives that favor biodegradable bioplastics over conventional fossil-based plastics. This action proposes targeted tax reductions for products made predominantly from certified biodegradable plastics, with the goal of narrowing the price gap between products made with conventional plastics and those made with biodegradable alternatives.

Design: This action would require: Legislation establishing tax reduction and scope. Clear regulation and enforcement, since many environmental laws fail at the implementation stage, including: i. eligibility criteria for tax incentives (e.g., minimum biodegradable content, verified biodegradability standards). ii. auditing mechanisms to prevent misuse, such as products labeled “bioplastic” that are bio-based but not biodegradable, or products containing only a small fraction of biodegradable material. iii. phased implementation, potentially starting with high-impact applications where replacing conventional plastics yields the greatest environmental benefit. Key actors include national and/or subnational governments, tax authorities, certification bodies, manufacturers opting into the program, and end users opting into buying products.

Assumptions: This action assumes that: Governments are willing and able to implement and enforce differentiated tax schemes. Traditional plastics resin producers will not lobby enough so as to stop the law. Price reductions at the product level are sufficient to meaningfully influence purchasing and adoption decisions. Certification systems can accurately distinguish between genuinely biodegradable materials and greenwashed alternatives.

Risks of Failure & “Success”: The policy could fail if enforcement is weak, allowing non-compliant products to benefit from incentives, or if administrative complexity discourages participation. Jurisdictional differences in taxation could also lead to production shifting across borders rather than reducing overall plastic harm. Even “successful” implementation may have unintended consequences, such as encouraging overconsumption of disposable products simply because they are labeled biodegradable, rather than reducing total plastic use.

Action 2: Large-scale public awareness campaigns on the human health impacts of traditional plastics

Purpose: Currently, public discourse around plastics focuses primarily on environmental damage, while the human health impacts of conventional plastics—such as chemical exposure, bioaccumulation, and endocrine disruption—remain relatively under-communicated. This limits public pressure for change and weakens demand for safer alternatives. This action proposes coordinated awareness campaigns that frame conventional plastics as a public health issue, similar to past campaigns against tobacco or excessive sugar consumption.

Design: This action would require: Global actors (e.g., WHO, UN agencies, international NGOs) to support and legitimize messaging at a global scale. National and local governments to adapt campaigns to local contexts and regulatory priorities. Startups and research institutions working on plastic alternatives to contribute evidence-based narratives and real-world solutions. Influencers, educators, and media organizations to amplify messages beyond traditional policy channels.

Assumptions: This action assumes that Increased awareness of health risks will meaningfully influence consumer behavior and political support. Also, that influencers and media actors can communicate complex health information responsibly. That global and local actins can be integrated.

Risks of Failure & “Success”: The campaign could fail if messages are oversimplified, sensationalized, or perceived as fear-based, leading to public distrust. There is also a risk of backlash from industry actors framing the campaign as anti-innovation or anti-consumer or leading to jobs reduction.

Action 3: Establish dedicated biopolymer research and governance centers at national and global levels

Purpose: At present, governance and reaseadrh and development of new biopolymers and engineered production strains is fragmented across regulatory agencies, academic labs, and industry actors. This fragmentation slows safe innovation and creates uncertainty around implementation, standards, and scale-up. This action proposes the creation of dedicated research and governance centers at national or subnational levels, complemented by a global network coordinating best practices and knowledge sharing.

Design: These centers would: i. Support regulatory implementation, including biosafety and material safety evaluation for new biopolymers. ii Conduct applied research on performance, degradation, and real-world applications of biodegradable plastics. iii. Serve as interfaces between academia, startups, industry, and regulators. iv. Participate in global networks to harmonize standards, share data, and reduce duplication of effort. Key actors include governments (as funders), universities, public research institutes, and international coordination bodies, as well as industry and startups.

Assumptions: This action assumes that: Governments are willing to fund long-term, interdisciplinary centers rather than short-term projects. Centralized expertise improves both safety and innovation outcomes. International collaboration is feasible despite differences in regulation and economic priorities.

Risks of Failure & “Success”: These centers could fail by becoming overly bureaucratic or disconnected from real industrial needs. They may also privilege dominant technological pathways, limiting diversity in approaches. A “successful” network might unintentionally centralize decision-making power, creating gatekeepers that slow innovation or disadvantage smaller players without access to these institutions.

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.

5. 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 expected impact and feasibility of the proposed actions, I would prioritize Action 1: tax incentives for biodegradable and bio-based plastics as the primary governance intervention.

The main reason is that cost remains the dominant barrier to large-scale adoption of biodegradable plastics, despite their availability and well-documented environmental benefits. Awareness campaigns (Action 2) and research centers (Action 3) already exist to some degree, and bioplastics are commercially available today. What has not been broadly or consistently implemented is a policy mechanism that directly and effectively reduces the price gap between fossil-based plastics and biodegradable alternatives through governmental incentives.

For a national or subnational policymaker audience (e.g., ministries of industry, environment, or finance), Action 1 offers the highest near-term leverage. In contrast, awareness campaigns rely on slower cultural change, and research centers operate on longer innovation timelines.

Action 3—the creation or reorientation of dedicated biopolymer research and governance centers—would be a strong second priority because will be important to enhance laws. Importantly, this does not necessarily require creating entirely new institutions. Existing research centers could be re-scoped through regulation or funding requirements.

Action 2 is expected to emerge organically once Action 1 is implemented. As economic incentives shift markets, industry groups, startups, researchers, and civil society actors are likely to amplify awareness efforts and public communication, especially around human health impacts. In this sense, Action 1 can act as a catalyst for the other two actions.

Trade-offs, assumptions, and uncertainties: This prioritization assumes that governments are willing to intervene in markets through fiscal policy and that tax incentives will translate into lower end-user prices. There is also uncertainty around political feasibility, especially in jurisdictions resistant to environmental taxation or subsidies.

Week 2 Prep: Homework Questions from Professor Jacobson: Error Rate: 1:106 Throughput: 10 mS per Base Addition basic error rate of DNA polymerase without proofreading is roughly 1 error per 10² to 10⁶ bases Biology resolves this discrepancy through multiple layers of fidelity control, including polymerase proofreading, mismatch repair systems, and cell-cycle checkpoints that prevent propagation of damaged DNA.

Average Human Protein: 1036 bp • Longest Human Proteins (PKS): >100kbp Since most amino acids are encoded by multiple synonymous codons, the number of possible DNA sequences that could encode the same protein is astronomically large. Not all work due to biological constrains: Codon usage bias: different organisms preferentially use certain synonymous codons, which affects translation efficiency and protein yield. mRNA structure: different nucleotide sequences form different local structures that affect ribosome binding and stability. Embedded regulatory elements: coding regions can contain splice sites, RNA-binding motifs, or secondary signals that influence expression.

Homework Questions from Dr. LeProust: phosphoramidite DNA synthesis.

it is difficult to synthesize oligos longer than ~200 nt Each nucleotide addition step has a small but non-zero error rate (incomplete coupling, deletions, or side reactions). As oligo length increases, these errors accumulate multiplicatively, leading to a rapidly decreasing fraction of full-length, error-free molecules. Beyond ~150–200 nt, yield and fidelity drop sharply, making purification inefficient and expensive.

A 2000 bp sequence would require ~2000 sequential synthesis steps, which would result in near-zero yield of full-length correct molecules due to accumulated errors. Instead, long genes are built by assembling shorter oligos (e.g., 50–200 nt) using enzymatic methods such as PCR-based assembly or Gibson assembly

Homework Question from George Church: 1. Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

lysine is universally essential across animal biology (not unique to any engineered or extinct species), to prevent organisms from surviving without an external lysine supply is not biologically plausible or unique—it simply reflects an existing nutritional requirement. This makes the fictional Lysine Contingency concept not a realistic.

Note on AI use: A large language model (ChatGPT) was used solely to assist with English grammar, spelling, and clarity of writing. All ideas, goals, policy actions, and arguments presented in this assignment were developed independently by the author and not generated by the language model.

Week 2 HW: DNA Read, Write, and Edit

Part 1: Benchling & In-silico Gel Art 🦠 Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks:

Part 3: DNA Design Challenge I chose poly(3-hydroxyalkanoate) polymerase / PHB synthase (PhaC) from Cupriavidus necator (UniProt accession P23608) because it is a key enzyme in microbial bioplastic production. PhaC catalyzes the polymerization of (R)-3-hydroxybutyryl-CoA monomers to form poly(3-hydroxybutyrate) (PHB), and engineered variants of PhaC are widely used to broaden substrate specificity and produce other polyhydroxyalkanoates (PHAs). I obtained the amino-acid sequence from UniProt (entry P23608) in FASTA format.

MA T G K G A A A S T Q E G K S Q P F K V T P G P F D P A T W L E W S R Q W Q G T E G N G H A A A S G I P G L D A L A G V K I A P A Q L G D I Q Q R Y M K D F S A L W Q A M A E G K A E A T G P L H D R R F A G D A W R T N L P Y R F A A A F Y L L N A R A L T E L A D A V E A D A K T R Q R I R F A I S Q W V D A M S P A N F L A T N P E A Q R L L I E S G G E S L R A G V R N M M E D L T R G K I S Q T D E S A F E V G R N V A V T E G A V V F E N E Y F Q L L Q Y K P L T D K V H A R P L L M V P P C I N K Y Y I L D L Q P E S S L V R H V V E Q G H T V F L V S W R N P D A S M A G S T W D D Y I E H A A I R A I E V A R D I S G Q D K I N V L G F C V G G T I V S T A L A V L A A R G E H P A A S V T L L T T L L D F A D T G I L D V F V D E G H V Q L R E A T L G G G A G A P C A A A A G L E L A N T F S F L R P N D L V W N Y V V D N Y L K G N T P V P F D L L F W N G D A T N L P G P W Y C W Y L R H T L P A E R A Q G T G Q A D R V R R A G G P G Q H R R P Y I Y G S R E D H I V P W T A A Y A S T A L L A N K L R F V L G A S G H I A G V I N P P A K N K R S H W T N D A L P E S P Q Q W L A G A I E H H G S W W P D W T A W L A G Q A G A K R A R P A N Y G N A R Y R A I E P A P G D T S K P R H

Although the genetic code is universal, most amino acids are encoded by multiple codons. However, different organisms do not use these synonymous codons at the same frequency. This phenomenon is known as codon bias. If a gene from one organism is expressed in a different host, the original codons may be rare in the new host, which can reduce translation efficiency, among other problems.

Codon optimization is necessary to modify the DNA sequence so that it uses codons preferred by the host organism. Optimizing codon usage can significantly increase protein expression levels, improve translation efficiency, enhance mRNA stability, and reduce the likelihood of misfolding or premature termination. For this project, I chose to optimize the phaC gene for Escherichia coli. I selected E. coli because it is the chassis organism we use at Bioplastix.

I used https://www.genscript.com/ as a code optimizattion tool (Job ID: 20260216015729685186).

At Bioplastix, the engineered bacteria produce the enzymes intracellularly. Under specific metabolic conditions, these enzymes catalyze the polymerization of monomers inside the cell, leading to intracellular accumulation of the biopolymer.

To achieve that, once the codon-optimized DNA sequence is obtained, it is inserted into a plasmid (an expression vector containing the necessary regulatory elements, such as a promoter and terminator). This plasmid is then be inserted into the host organism, Escherichia coli, for protein production.

In such a cell-dependent system, the DNA sequence is transcribed into messenger RNA (mRNA) by RNA polymerase. The mRNA is then translated by ribosomes into the PhaC protein. Depending on the design of the genetic construct, the gene can be placed under a constitutive promoter, where the protein is continuously produced, or under an inducible promoter, where expression is triggered by specific conditions (such as the presence of IPTG or oxigen).

Alternatively, the protein could also be produced using a cell-free expression system, where the DNA is added to a reaction mixture containing ribosomes, tRNAs, nucleotides, and enzymes necessary for transcription and translation. This allows protein production without living cells, although large-scale industrial production until now typically relies on cell-based systems.

Part 4: Prepare a Twist DNA Synthesis Order

Link Sharing From Benchling: https://benchling.com/s/seq-l6sbaCHItMO0QD5lHD4M?m=slm-38MASpmHAGVSi3hySqfU

Part 5: DNA Read/Write/Edit 5.1 DNA Read (i) What DNA would you want to sequence (e.g., read) and why? I would like to sequence DNA from environmental microbial communities, particularly from diverse and extreme environments such as soil, marine ecosystems, open dumps, and high-stress habitats. These environments often contain microorganisms with unique metabolic capabilities.

By sequencing DNA from environmental samples (metagenomic sequencing), we could identify novel enzymes involved in polymer biosynthesis, including new variants of PHA synthases or related polymerizing enzymes. These enzymes may have improved catalytic efficiency, altered substrate specificity, or greater stability under industrial conditions. Discovering new enzymes through DNA sequencing could enable the development of more efficient bioplastic production systems and potentially allow the synthesis of novel polymers with enhanced material properties.

(ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why? I would use Illumina sequencing technology, a second-generation (next-generation sequencing, NGS) platform. Illumina sequencing is well-suited for metagenomic analysis because it provides high-throughput, highly accurate short reads at relatively low cost, making it ideal for sequencing complex microbial communities. It is second generation because it performs massively parallel sequencing, requires DNA amplification (cluster generation), and sequences millions of fragments simultaneously.

The input is extracted environmental DNA (metagenomic DNA) from microbial communities.

Sample preparation steps:

1. DNA extraction from environmental samples (soil, water, etc.)
2. Fragmentation of DNA into smaller pieces (typically ~200–500 bp)
3. End repair and A-tailing
4. Adapter ligation (short known sequences attached to fragment ends)
5. PCR amplification to enrich properly ligated fragments
6. Library quantification and quality control

Illumina sequencing works as follows:

1. The prepared DNA library is loaded onto a flow cell.
2. Fragments bind to complementary oligos attached to the flow cell.
3. Bridge amplification creates clusters of identical DNA fragments.
4. Fluorescently labeled reversible terminator nucleotides are added.
5. During each cycle, one nucleotide is incorporated per cluster.
6. A camera detects the fluorescent signal emitted by the incorporated base.
7. The terminator is cleaved, and the next cycle begins.

Each nucleotide (A, T, C, G) carries a different fluorescent signal. The imaging system detects the color emitted at each cycle, and software converts fluorescence intensity into base calls (A, T, C, or G).

5.2 DNA Write

The DNA I would want to “write” (synthesize) would be a set of candidate polymerizing enzymes identified from the environmental metagenomic library, especially novel PHA synthase (PhaC-like) genes or other enzymes predicted to catalyze polymer formation. The main reason to synthesize these genes is to rapidly test them in a standardized host. I would order codon-optimized versions of each candidate enzyme gene for Escherichia coli, because E. coli is the chassis organism we use at Bioplastix.

I would use solid-phase phosphoramidite DNA synthesis, the standard chemical DNA synthesis technology used by companies such as Twist Bioscience. In the past we already used Twist for synthesis of new enzymes. It is very useful because enables codon optimization, Introduction of specific mutations, Removal of unwanted restriction sites, parallel synthesis of multiple variants.

With this methodd DNA is synthesized one nucleotide at a time on a solid support using phosphoramidite chemistry. This produces short DNA fragments (typically 150–200 bp). Since genes are longer than individual oligos, overlapping oligonucleotides are assembled into full-length genes using: PCR-based assembly, Gibson Assembly or Enzymatic ligation methods. The assembled gene is amplified and sometimes enzymatically corrected to reduce synthesis errors. The final construct is cloned into a plasmid and verified by DNA sequencing before delivery.

From a practical perspective at Bioplastix, the main limitation of outsourcing DNA synthesis to companies such as Twist Bioscience is cost. Synthesizing long gene sequences can be expensive, particularly when testing multiple enzyme variants. Each synthesis order costs approximately $1,500 USD. After synthesis, there are further costs associated with Transforming it into our production strains and Screening and validating expression. Overall, with a typical budget, we are able to test approximately 5–10 new enzyme candidates per round.

5.3 DNA Edit The DNA I would want to edit is the genome of our production strain (e.g., Escherichia coli) in order to improve its efficiency as a bioplastic-producing chassis organism. Specifically, I would focus on :

1️⃣ Expanding substrate utilization: One of our current priorities is enabling the strain to efficiently consume alternative and low-cost carbon sources, particularly sucrose. Editing the genome to introduce or optimize sucrose transporters and metabolic pathways would allow the bacteria to convert a wider range of feedstocks into biopolymer, improving economic feasibility and sustainability.

2️⃣ Increasing carbon flux toward polymer production: I would edit genes involved in central carbon metabolism to redirect more carbon toward the PHA biosynthesis pathway.

3️⃣ Engineering polymerizing enzymes: I would also edit the genes encoding polymerizing enzymes (such as PhaC) to Increase substrate affinity, Improve catalytic efficiency and Enhance thermostability. Thermotolerant enzymes would be particularly valuable at industrial scale, where higher fermentation temperatures reduce cooling costs and contamination risk.

To perform these genome edits, I would use CRISPR-Cas9 genome editing, combined with homologous recombination. CRISPR-based systems are precise, programmable, and highly efficient, making them ideal for metabolic engineering in bacterial systems. CRISPR-Cas9 uses: i. A guide RNA (gRNA) designed to match a specific DNA target sequence. ii. The Cas9 nuclease, which creates a double-strand break at the target site

After the break is introduced, the cell repairs the DNA. If a repair template is provided (homology-directed repair, HDR), specific edits such as insertions, deletions, or point mutations can be introduced.

Limmitations include Metabolic burden (Multiple edits can stress the cell and reduce growth rate) and Regulatory complexity (Metabolic networks are highly interconnected; editing one pathway may produce unexpected downstream effects).

Week 3 HW: Lab Automation

Python Script for Opentrons Artwork: https://colab.research.google.com/drive/14m54uLCM5UtsggVjU2Ucxh5hhtNELWD2#scrollTo=pczDLwsq64mk&line=4&uniqifier=1

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

I found the paper ¨Opentrons for automated and high-throughput viscometry¨ very interesting.

¨The operating protocol involves measuring the amount of liquid dispensed over a set time for given dispense conditions. Data collected at different set dispense flow rates was used to train an ensemble machine learning regressor to predict Newtonian liquid viscosity¨. They demonstrated the ability of the proxy viscometer to characterize the rheological behavior of two types of power-law fluids.

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

   For my final project, I intend to focus on a challenge that is directly relevant to my startup, Bioplastix, which develops technologies for the production of bio-based copolymers with plastic properties. I am particularly interested in exploring how low-cost automation tools, such as the Opentrons OT-2, can accelerate enzyme discovery and biopolymer development workflows. I have two potential project directions:

Idea 1: High-Throughput Screening of Polymerizing Enzymes: Bioplastix has access to a diverse environmental enzyme library. Our goal is to identify enzymes capable of polymerizing specific monomers into useful copolymers. An initial in silico screening step can be performed by identifying genetic sequences that are homologous to known polymerizing enzymes. However, the key bottleneck occurs during experimental validation in the wet lab. Using automation tools, we could perform parallel screening of up to 96 enzyme candidates per plate, standardizing reaction setup to reduce variability. This will be possible Implementing colorimetric assays to detect polymer formation. The automation platform would allow us to screen dozens (or hundreds) of enzymes under consistent conditions, significantly accelerating discovery while reducing human error.

Idea 2: Automated Cell-Free Production of Copolymers: A second direction would be to explore automated workflows for cell-free production systems. Instead of expressing enzymes in living cells, we could use cell-free systems to produce polymers. This would involve screen different reaction conditions (pH, cofactors, substrate ratios, temperature), optimize copolymer production in a high-throughput format.

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project