Week 2 Lecture Preparation Assignment

1. What is the error rate of DNA polymerase?

• Nature’s biological machinery for copying DNA, error-correcting polymerase, has an error rate of approximately 1:10^6 (one in a million).
• This compares to the human genome length of approximately 3 billion base pairs (3 Gb).
• To handle the discrepancy between this error rate and the vast size of the genome, biology utilizes multiple proofreading mechanisms, including 5’-3’ error-correcting exonucleases and 3’-5’ proofreading exonucleases during template-dependent primer extension

2. Coding for a Human Protein

• For an average human protein (approximately 1,036 bp or 345 amino acids), there are an enormous number of DNA sequences that can code for it due to the redundancy of the genetic code, where multiple codons often translate to the same amino acid.
• In practice, many of these potential codes do not work because of technical “complexities” that hinder synthesis and biological function, such as extreme GC content (local content ≥90% or ≤10%), hairpins and secondary structures, long repeats (tandem or inverted), and homopolymers (runs of an identical base longer than 30 bp)

1. What is the most commonly used method for oligo synthesis today?

The dominant method is solid-phase phosphoramidite chemistry, which synthesizes DNA one nucleotide at a time in the 3’ → 5’ direction.

2. Why is it difficult to synthesize oligos longer than ~200 nt?

Direct oligonucleotide synthesis becomes increasingly difficult as sequence length increases because each synthesis cycle introduces a small probability of error. As the number of base additions grows, these errors accumulate, leading to an exponential decrease in yield. In practice, this means that truncated products and incorrect sequences begin to dominate the final mixture.

Beyond approximately 200 nucleotides, the fraction of full-length, sequence-correct oligos becomes very low. This is a direct consequence of step-wise coupling inefficiency: even when individual base addition steps are highly efficient, the cumulative probability of producing a perfect sequence decreases exponentially with length. For example, if each coupling step has a small failure rate, the overall yield after N steps follows an exponential decay curve (often approximated by (1−1/N)^N ≈ 37%), resulting in a dramatic loss of high-quality, full-length DNA as oligo length increases.

3. Why can’t a 2000 bp gene be made via direct oligo synthesis?

A 2,000 bp gene cannot be produced via direct oligonucleotide synthesis because the cumulative yield loss across thousands of coupling cycles would result in an effectively zero number of sequence-perfect molecules. As synthesis length increases, small inefficiencies at each base addition compound exponentially, making full-length, error-free products exceedingly rare.

As a result, direct synthesis of long genes would lead to extremely low yields of full-length DNA, high error rates, and prohibitive costs. Instead, long genes are constructed by assembling shorter, sequence-verified oligonucleotides or gene fragments using methods such as enzymatic assembly, PCR-based assembly, or Gibson Assembly, which allow errors to be corrected at intermediate steps and dramatically improve overall fidelity.

Question Chosen

What are the 10 essential amino acids in animals, and how does this affect the “Lysine Contingency”?

The 10 Essential Amino Acids

Animals cannot synthesize these amino acids and must obtain them from their diet:

• Histidine
• Isoleucine
• Leucine
• Lysine
• Methionine
• Phenylalanine
• Threonine
• Tryptophan
• Valine
• Arginine (essential in many animals, especially during growth)

Implications for the Lysine Contingency

Lysine’s essentiality creates a biological dependency that can act as a containment strategy. Organisms engineered to require external lysine supplementation would be unable to survive outside controlled environments.

This reinforces the idea that metabolic dependencies can be used as safety mechanisms, embedding governance directly into biological design rather than relying solely on external regulation.

Program Chosen: BioStabilization Systems (BoSS)

Proposed Concept: Nature-Inspired Modular Biostabilization for Cell Therapies

The BoSS program addresses a critical bottleneck in modern medicine: the extreme fragility of biologics and cell therapies and their dependence on an expensive, failure-prone cold chain. I propose a modular biostabilization strategy inspired by naturally stress-tolerant organisms, combined with scalable materials engineering.

Core Idea

Develop a hybrid intracellular–extracellular stabilization platform that enables long-term room-temperature storage of cell therapies by combining:

1. Intracellular protection

   • Transient expression or loading of protective molecules inspired by anhydrobiotic organisms (e.g., trehalose analogs, intrinsically disordered stress proteins).
   • These molecules stabilize membranes and proteins during dehydration and rehydration cycles without permanently altering cellular identity.

2. Extracellular engineered matrices

   • Encapsulation of cells within biocompatible, glass-like polymer matrices that mimic vitrification without freezing.
   • Materials engineered to reversibly dissolve upon rehydration, enabling rapid cell recovery at the point of care.

Technical Approach

• Screen and optimize protective molecules using high-throughput assays for post-rehydration viability and function.
• Engineer scalable cell-processing workflows compatible with existing biomanufacturing pipelines.
• Design restoration protocols that ensure rapid functional recovery without specialized infrastructure.

Why This Fits BoSS

This approach directly addresses both BoSS technical areas:

• Cell interventions for stabilization and restoration
• Scalable processing systems for deployment and commercialization

By eliminating ultra-cold storage requirements, this system would dramatically reduce costs, improve resilience to supply chain disruptions, and expand access to advanced biologics in remote and resource-limited settings.

Broader Impact

If successful, room-temperature biologics would:

• Enable stockpiling of life-saving therapies
• Reduce product loss due to logistics failures
• Democratize access to advanced medicine beyond major medical centers

This proposal treats stability not as a logistical afterthought, but as a design constraint embedded directly into biological systems, aligning with BoSS’s vision of transformative, nature-inspired biotechnologies.