Week 1 HW: Principles and Practices

Application: Bio-Ink decentralized DNA System

The concept: I want to develop a Desktop DNA Printer- a high- fideliyy, benchtop enzymatic synthesis tool that allows researchers to “print” long. Complex strands of DNA overnight without relying on third-party vendors

Why? Current DNA synthesis is centralized. If you’re researcher in a resource-limeted setting of a fast-moving startup, waiting 2-4 weeks for a shipment from a centeral hub slows down the “ Design-Build-Test” cycle. This tool would democratize genetic engeneering, making it as accessible as 3Dprinting is for mechanical engeneers

Governance & Policy Goals To ensure this tool contributes to an ethical future, we must balance open science with global security.

Goal 1: Non – Malfeasance ( Biosecurity) Sub-goal A: Proactive sequence Screening. Prevent the synthesis of known regulated phatogens or high toxic proteins Sub-Goal B : Anti-Obfuscation. Ensure that :broken up” sequences ( small fragments that could be assembled into a phatogen ) are detected even when printed across different sessions Goal 2 : Equaty & Access Subgoal A: Geographic Neutrality. Enshuring the hardware and “ink”(reagents) aren’t restricted only to welthy nations. Sub-goal B: Open-Source Interoperability, Preventing “vendor lock-in” where only one company controlls the “digital DNA’’ ecosystem. Aspect,Action 1: Technical (Cloud-Gating),Action 2: Incentive (Trusted Researcher Tier),Action 3: Regulatory (Bio-Liability Insurance) Purpose,“Moves from voluntary screening to an ““Internet Handshake”” requirement before the printer fires.”,“Creates a ““Fast Track”” for vetted labs, similar to ““TSA PreCheck”” for scientists.”,“Requires users to carry liability insurance to purchase reagents, shifting risk to the private market.” Design,“Actors: Manufacturers & Cloud Providers. The printer won’t work without an encrypted connection to a centralized ““Red Flag”” database.”,“Actors: Academic Institutions & Federal Regulators. Labs undergo a one-time vetting; in exchange, they get less intrusive screening.",Actors: Insurance Companies & Law Enforcement. Insurers audit lab safety protocols before granting a policy. Assumptions,“Assumes users won’t ““jailbreak”” the hardware or find ways to run it offline.”,“Assumes ““vetted”” individuals cannot be radicalized or coerced (the ““insider threat””).",Assumes insurance companies have the technical expertise to actually assess bio-risk. Risks,Failure: Hackers bypass the cloud gate. Success Risk: Research in remote areas with poor internet is stifled.,“Failure: Creates a ““caste system”” where small startups are stuck in the slow lane.”,“Failure: High premiums kill ““garage biology”” and small-scale innovation.”

Target Audiance: The International Gene Synthesis Consortium (IGSC) and National Ministies of science Priority: I recomend a Hybrid Model of Action 1 and Action 2

Why? Pure technical restriction is teh most effectiv way to prevent a “bad actor” from printing a toxin on day one. Hoever, it creates a massive burden for legitimate scientists.By introducing the “Trusted Researcher” Tier, we create a relife valve. If a lab is verred by theyr university and a federal agency, their “Cloud-Gate” becomes a " Soft-Gate” faster processing and more privacy for their sequencing. Trade-offs and Incertenties: The black market trade of: By making legal machines highly regulated, we might inadvertently create a market for “jailbroken” or non-complaint printers manufactured in juristrictions with zero oversight The Privecy Uncertenty: We are assuming researchers will be ok with a “Big Brother” system seeing what they point. In rea;ity, the feare of intellectual property theft might lead to significant pushback from the private sectore

Homework 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? Polymerase error rate: 1:10^6 The human genome has 4.2 billion base pairs, which means that polymerase makes 3200 errors at each celll division. In order to deal with this high error rate, cells have systems made of multiple proteins that correct those errors, although they are not perfect. 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? Human protein: ~1036bp –> ~345 amino acids W Different codons can enconde for the same AA, but not all these sequences are equally efficcinet. Depending on the organism, a codon might be better for correctly and fast/slow enough being translated into an amino acid. Homework Questions from Dr. LeProust: What’s the most commonly used method for oligo synthesis currently? The solid-phase phosphoramidite chemical synthesis Why is it difficult to make oligos longer than 200nt via direct synthesis? error accumulation and chemical limitations Why can’t you make a 2000bp gene via direct oligo synthesis? mailny due to error accumulation Homework 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 that animals cannot synthesize on their own and must obtain through their diet are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, Valine

The “Lysine Contingency” refers to the fictional failsafe mechanism in Michael Crichton’s Jurassic Park, where resurrected dinosaurs were genetically engineered to lack the ability to produce lysine, one of these essential amino acids. The idea was that without lab-supplied lysine supplements, the dinosaurs would die, preventing them from surviving if they escaped containment. Lysine is an essential amino acid for all animals which reinforces why this contingency is fundamentally flawed as a control measure. In reality, no animal can synthesize lysine endogenously—they all rely on dietary sources like plants, insects, or meat, which are abundant in natural ecosystems. If the engineered dinosaurs escaped, they could simply consume lysine-rich foods in the wild (as they do in the story), rendering the dependency moot. It highlights a clever narrative device but poor fictional science, as it overlooks basic nutritional biology and ecology. A more effective contingency might target a non-essential amino acid or something truly unique to the lab environment.

Professor Jacobson1. DNA Polymerase Error Rate and Biological CorrectionError Rate: The intrinsic error rate of DNA polymerase is approximately $1:10^{6$ (one mistake for every million bases).Comparison to Genome: The human genome contains roughly 3 billion ($3 \times 10}9$) base pairs. Without correction, this would result in ~3,000 errors every time a cell divides.Biological Solutions: * Proofreading: Polymerase has a self-correcting 3’ to 5’ exonuclease activity that “snips” out incorrect bases. This improves accuracy to about $1:10^{9$.Mismatch Repair: After replication, secondary protein systems scan the DNA to fix remaining discrepancies.Genomic Buffer: Much of the human genome is non-coding (introns) or “dormant,” meaning many mutations occur in areas that don’t affect cell function.Apoptosis: If a mutation is too severe (common in cancer-prone cells), the cell is programmed to undergo “cell suicide.“2. Protein Coding Diversity and LimitationsTheoretical Ways to Code: For an average human protein (~345 amino acids / 1036 bp), there are roughly $10}{157}$ possible DNA sequences that could code for it, because most amino acids are represented by multiple codons.Practical Limitations:Codon Bias: Different organisms prefer specific codons. “Rare” codons can slow down translation because the corresponding tRNA is in short supply.mRNA Folding: The DNA sequence dictates how the resulting mRNA folds. High-stability folds can stall ribosomes or cause premature termination of the protein chain.Dr. LeProust (Oligo Synthesis)1. Current Synthesis MethodThe gold standard is solid-phase phosphoramidite chemical synthesis.2. The 200nt LimitSynthesizing strands longer than 200 nucleotides (nt) is difficult due to stepwise error accumulation.Even with a 99.5% efficiency rate per step, the mathematical probability of a perfect sequence drops exponentially as length increases.By 200 bases, the yield of the “perfect” product is very low. Additionally, side reactions and physical crowding on the synthesis support hinder the process.3. Why 2000bp is Impossible via Direct SynthesisA 2000bp gene (4000 total nucleotides) cannot be made in one go because the cumulative error rate would result in zero functional product. Instead, scientists synthesize small “oligos” and then stitch them together using biological assembly methods.George Church1. The 10 Essential Amino AcidsThe ten amino acids that most animals (including humans) cannot produce on their own are:Arginine (R)*, Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Threonine (T), Tryptophan (W), and Valine (V).*Note: Arginine is often considered “semi-essential,” especially for growth.2. View of the “Lysine Contingency"The “Lysine Contingency” (from Jurassic Park) is a flawed scientific concept for two main reasons:Vertebrate Biology: No vertebrate can synthesize lysine naturally. Therefore, “knocking out” the ability for dinosaurs to make it doesn’t change anything—they were already dependent on their diet for it.Ecological Reality: In the wild, lysine is abundant in plants and other animals. If the dinosaurs escaped, they would simply eat lysine-rich food (like soy or meat), rendering the laboratory “failsafe” completely useless.