Week 1: Professor Questions

Answer

Executive Summary:
DNA polymerase intrinsic error rate (~10⁻⁷) would cause ~320 errors per human genome replication (3.2 × 10⁹ bp). Biology employs multilayer error correction (proofreading, mismatch repair, excision repair) to achieve final fidelity of ~10⁻⁹ to 10⁻¹⁰ errors per base per division, yielding 0.3-3 errors per replication in normal somatic cells.

Error Rate of DNA Polymerase

DNA polymerase has an intrinsic error rate of approximately 1 error per 10⁷ nucleotides during DNA synthesis. With integrated 3’ to 5’ exonuclease proofreading activity, this improves to approximately 1 error per 10⁸-10⁹ nucleotides.

Comparison to Human Genome Length

The human genome contains approximately 3.2 × 10⁹ base pairs.

Without proofreading:

• Error rate: ~10⁻⁷ per nucleotide
• Expected errors per replication: ~320 errors per genome copy

With proofreading:

• Error rate: ~10⁻⁸ to 10⁻⁹ per nucleotide
• Expected errors per replication: ~3-32 errors per genome copy

How Biology Deals with This Discrepancy

Biology employs multiple layers of error correction that act sequentially:

1. Proofreading (3’ → 5’ exonuclease activity)

   • DNA polymerase detects incorrect base pairing via geometric distortion
   • Removes mismatched nucleotide immediately
   • Reduces error rate by approximately 100-1000-fold

2. Mismatch Repair (MMR) System

   • Post-replication surveillance mechanism
   • In bacteria (E. coli): MutS, MutL, and MutH proteins
   • In eukaryotes: MSH (MutS homolog), MLH (MutL homolog), and PMS protein families
   • System identifies mismatched base pairs, excises incorrect strand segment, and resynthesizes
   • Further reduces error rate by approximately 100-1000-fold

3. Nucleotide Excision Repair (NER)

   • Repairs bulky DNA lesions (UV-induced thymine dimers, chemical adducts)
   • Removes damaged nucleotide segments (20-30 nt patches)

4. Base Excision Repair (BER)

   • Corrects small base modifications (deamination, oxidation, alkylation)
   • DNA glycosylases remove damaged bases; AP endonucleases process abasic sites

Result:
The combined fidelity of replication in eukaryotic somatic cells typically achieves ~10⁻⁹ to 10⁻¹⁰ errors per base per cell division, depending on organism, cell type, and proliferation status. This ensures 0.3-3 errors per genome replication under normal physiological conditions.

Note: Fidelity varies by context. Cancer cells with MMR defects exhibit 100-1000× higher mutation rates. Germline cells employ additional proofreading mechanisms. Some DNA polymerases (e.g., Pol η, translesion synthesis polymerases) have lower fidelity by design for specialized repair functions.

Answer

Executive Summary:
For a typical 400-residue protein, the number of synonymous DNA sequences (due to codon degeneracy) is astronomically large—on the order of 10¹⁰⁰ or more, calculated as the product of synonymous codon counts across all positions. In practice, most sequences fail due to codon usage bias, mRNA secondary structure, RNA instability, splicing interference, cryptic regulatory elements, and synthesis/cloning constraints.

Number of Different Ways to Code for a Protein

The genetic code is degenerate—61 sense codons encode 20 standard amino acids plus start/stop signals. Each amino acid (except Met and Trp) has multiple synonymous codons:

• Leucine, Serine, Arginine: 6 codons each
• Isoleucine: 3 codons
• Methionine, Tryptophan: 1 codon each

For an average human protein (~400 amino acids):

The total number of synonymous DNA sequences is the product of synonymous codon counts across all positions:

N = ∏(i=1 to 400) n_i

where n_i = number of synonymous codons for amino acid i.

Rough estimate:

• Average degeneracy per amino acid ≈ 3 codons (weighted by frequency)
• Total combinations ≈ 3⁴⁰⁰ ≈ 10¹⁹⁰ possible DNA sequences

Even conservative estimates (e.g., leucine-rich proteins) yield 10¹⁰⁰+ combinations.

Why All These Different Codes Don’t Work in Practice

Even though multiple sequences encode the same amino acid sequence, the vast majority fail to express functional protein due to:

1. Codon Usage Bias

• Each organism has preferred codons reflecting tRNA abundance (Plotkin & Kudla 2011)
• E. coli prefers different codons than humans (e.g., AGG/AGA rare in bacteria, common in mammals)
• Rare codons → ribosome stalling → may alter co-translational folding kinetics
• Using non-optimal codons can reduce expression 10-1000-fold (Gustafsson et al. 2004)

2. mRNA Secondary Structure

• Certain nucleotide sequences form stem-loops or hairpins
• Strong secondary structures can:
  • Block ribosome binding
  • Stall translation
  • Trigger mRNA degradation

3. RNA Stability

• AU-rich sequences → rapid mRNA degradation
• GC-rich sequences → more stable mRNA
• Wrong codon choice can drastically reduce mRNA half-life

4. Splicing Interference

• Certain sequences create cryptic splice sites
• Can cause exon skipping or intron retention
• Results in truncated or non-functional protein

5. Ribosome Binding Sites (RBS) Interference

• Shine-Dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes)
• Internal RBS-like sequences can cause premature translation initiation
• Results in truncated proteins

6. Restriction Enzyme Sites

• Cloning often requires avoiding certain restriction sites
• Limits sequence choices for practical molecular biology

7. Repetitive Sequences

• Long homopolymer runs (e.g., AAAAAA) cause synthesis/sequencing errors
• Can trigger recombination or replication errors

Quantitative Example: For a 10-amino acid peptide (assuming average 3-fold degeneracy), there are theoretically 3¹⁰ ≈ 59,000 synonymous sequences. However, accounting for all the constraints listed above, only an estimated 10²-10³ sequences (~1-2%) would be practically functional.

Answer

Executive Summary:
Phosphoramidite chemistry on solid-phase support (Caruthers method, 1981) is the current industry standard, with typical coupling efficiency of 98.5-99.5% per cycle and practical length ceiling of 150-200 nucleotides.

Phosphoramidite Chemistry (Solid-Phase Synthesis)

The phosphoramidite method on solid support is the dominant technology for oligonucleotide synthesis worldwide.

Key Features:

• Invented: Marvin Caruthers and colleagues (1981)
• Platform: Solid-phase synthesis on controlled-pore glass (CPG) or polystyrene beads
• Direction: 3’ → 5’ synthesis (chain grows from 3’-OH to 5’ end)
• Cycle efficiency: Typically 98.5-99.5% per nucleotide addition
• Practical length limit: 150-200 nucleotides for routine synthesis

Four-Step Cycle:

1. Detritylation (acid treatment)

   • Removes DMT (dimethoxytrityl) protecting group from 5’-OH
   • Exposes reactive hydroxyl for next nucleotide

2. Coupling (phosphoramidite addition)

   • Protected phosphoramidite monomer + tetrazole activator
   • Forms phosphite triester linkage
   • ~98-99.5% coupling efficiency

3. Capping (acetic anhydride)

   • Blocks unreacted 5’-OH groups
   • Prevents deletion sequences

4. Oxidation (iodine/water)

   • Converts unstable phosphite (P³⁺) to stable phosphate (P⁵⁺)
   • Forms phosphate backbone

Advantages:

• High throughput (96-384 well formats)
• Automated
• Scalable (nmol to µmol scale)
• Well-established chemistry

Current Platforms: Commercial platforms include BioAutomation and ABI/Applied Biosystems synthesizers for traditional column-based synthesis. Newer high-throughput approaches include Twist Bioscience (silicon-based microarray synthesis) and Custom Array (electrochemical synthesis on chips).

Answer

Executive Summary:
Cumulative coupling inefficiency (even at 99% per cycle) yields only ~13% full-length product at 200 nt. Dominant failure modes are deletion sequences from incomplete coupling, depurination during detritylation, and increasing purification difficulty as n-1, n-2… products accumulate.

Cumulative Coupling Errors and Deletion Sequences

The primary challenge is imperfect coupling efficiency in each phosphoramidite addition cycle.

The Mathematics of Error Accumulation:

• Coupling efficiency per cycle: typically 98.5-99.5%
• Stepwise failure rate: 0.5-1.5% per cycle
• Yield of full-length product = (coupling efficiency)^n where n = oligo length

Yield Calculation:

At 200 nucleotides with 99% efficiency:

• Only 13% of molecules are full-length correct sequence
• 87% are deletion products (n-1, n-2, n-3… truncations)

Specific Problems Beyond 200nt (in order of impact):

1. Deletion Sequences from Incomplete Coupling

   • Failed coupling at position i → all subsequent additions build on truncated chain
   • Creates heterogeneous mixture of n-1, n-2, n-3… products
   • Capping step blocks these from extending, but they remain in final pool

2. Depurination During Acid Treatment

   • Detritylation uses trichloroacetic acid or dichloroacetic acid
   • Causes glycosidic bond cleavage at purines (A, G)
   • Cumulative damage over 200+ cycles
   • Results in abasic sites and chain breaks

3. Purification Difficulty

   • Full-length (200 nt) vs. n-1 (199 nt) differ by <0.5% in mass
   • HPLC and PAGE separation becomes marginal
   • Impure product affects downstream applications

4. Secondary Structure Formation

   • Long single-stranded oligos form intramolecular hairpins during synthesis
   • Blocks reagent access to growing 3’-OH end (on solid support, growing from 3’ end)
   • Reduces effective coupling efficiency in later cycles

5. Synthesis Time and Cost

   • 200 cycles × 10-15 min/cycle = 33-50 hours continuous synthesis
   • Reagent consumption scales linearly
   • Low yields require larger scale synthesis → higher cost

Practical Solutions: Modern approaches avoid direct synthesis beyond 200 nt by using gene assembly from overlapping 60-80 nt oligos (polymerase cycling assembly, Gibson assembly), column-based assembly methods (e.g., Twist Bioscience chip synthesis followed by assembly), or emerging enzymatic synthesis using terminal deoxynucleotidyl transferase-based methods.

Answer

Executive Summary:
Direct phosphoramidite synthesis of 2000 nt is practically infeasible due to vanishingly low yields (0.99^2000 ≈ 10⁻⁹), prohibitive synthesis time (~2-3 weeks continuous), cumulative depurination, and insurmountable purification challenges. Modern gene synthesis uses hierarchical assembly of 60-80 nt oligos into fragments, then full-length genes.

Practical Infeasibility with Current Phosphoramidite Chemistry

Making a 2000 bp gene via direct oligonucleotide synthesis is practically infeasible with standard phosphoramidite chemistry due to insurmountable yield, time, and purification barriers.

Yield Barriers:

At 99% coupling efficiency (best-case scenario):

• Yield = 0.99^2000 ≈ 2 × 10⁻⁹ (0.0000002%)
• To obtain 1 picomole of full-length product requires ~0.5 moles of starting material
• Equivalent to ~660 grams of protected nucleotide phosphoramidites
• Material cost alone: ~$500,000 - $1,000,000

Even at 99.5% efficiency (exceptional, rarely achieved):

• Yield = 0.995^2000 ≈ 5 × 10⁻⁵ (0.005%)
• Still economically and practically prohibitive

Physical/Chemical Barriers:

1. Synthesis Time

   • Typical cycle time: 10-15 minutes per nucleotide addition
   • 2000 cycles = 20,000-30,000 minutes = 14-21 days continuous synthesis
   • Reagent degradation over extended periods
   • Instrument reliability over multi-week runs

2. Cumulative Depurination

   • 2000 acid detritylation steps
   • Each cycle causes low-frequency glycosidic bond cleavage at purines
   • Accumulates to extensive abasic sites and strand breaks

3. Secondary Structure Collapse

   • Long single-stranded DNA forms extensive intramolecular structure
   • Hairpins and G-quadruplexes block reagent access
   • Synthesis typically stalls beyond 300-400 nt even with optimized conditions

4. Solubility and Handling

   • Very long oligos can precipitate on solid support
   • Reduced accessibility to coupling reagents
   • Cleavage and deprotection become inefficient

Practical Solution: Hierarchical Gene Assembly

Modern commercial gene synthesis uses multi-step assembly:

Step 1: Oligo Synthesis

• Synthesize 30-50 oligonucleotides (60-80 nt each, with 20-40 nt overlaps)
• Yield per oligo: 60-95% (high quality)

Step 2: Fragment Assembly

• Assemble oligos into 4-6 intermediate fragments (400-600 bp each)
• Methods: Polymerase cycling assembly (PCA), Gibson assembly, Golden Gate
• Yield per fragment: 70-90%

Step 3: Final Assembly

• Combine fragments into full 2000 bp gene
• Gibson assembly or restriction enzyme-based methods
• Final yield: 60-85% overall

Example for 2000 bp gene:

• 40 oligos × 70 nt average = 2800 nt synthesized capacity
• Assemble into 5 fragments (~400 bp each)
• Final Gibson assembly into 2000 bp construct
• Overall yield: ~70% (vs. 10⁻⁹% for direct synthesis)

Commercial Gene Synthesis: Major vendors (Twist Bioscience, IDT, GenScript, Thermo Fisher) offer typical academic pricing of $0.07-0.20/bp, though this is highly variable depending on sequence complexity (GC content, repeats, secondary structure), turnaround time (5-10 days standard, 2-3 days expedited), and order volume. Standard turnaround is 5-10 days with rush options of 2-3 days.

(I chose this question from the three options)

Answer

Executive Summary:
The commonly listed essential amino acids in vertebrates include His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, and conditionally Arg. The “Lysine Contingency” from Jurassic Park is scientifically flawed because lysine is already naturally essential in all vertebrates—the genetic modification provides zero additional biocontainment. Moreover, lysine is abundant in all natural food sources, and deficiency takes months to years to be lethal.

The Commonly Listed Essential Amino Acids in Vertebrates

Essential amino acids cannot be synthesized de novo by vertebrate metabolism and must be obtained from diet. The standard list for humans and most vertebrates includes: Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K) [focus of Jurassic Park scenario], Methionine (Met, M), Phenylalanine (Phe, F), Threonine (Thr, T), Tryptophan (Trp, W), Valine (Val, V), and Arginine (Arg, R), which is conditionally essential—essential in juveniles, young/growing animals, and during illness, though adults can synthesize limited amounts via the urea cycle.

Mnemonic: “PVT TIM HALL” (Phe, Val, Thr, Trp, Ile, Met, His, Arg, Leu, Lys)

Note: The classification varies slightly by species and life stage. Arginine is typically considered semi-essential or conditionally essential in adult mammals.

The “Lysine Contingency” from Jurassic Park

In Jurassic Park (Michael Crichton, 1990), InGen implemented a “Lysine Contingency” as a biocontainment measure. The plan involved genetically engineering dinosaurs unable to synthesize lysine, making them dependent on lysine supplements in their food. The theory was that if they escaped, they would die from lysine deficiency. As Dr. Wu stated: “The lysine contingency is intended to prevent the spread of the animals is case they ever got off the island.”

Why the Lysine Contingency is Scientifically Flawed

Critical Problem: ALL ANIMALS ALREADY REQUIRE DIETARY LYSINE

1. Lysine is Naturally Essential in All Vertebrates

Humans, dinosaurs, birds, and mammals cannot synthesize lysine de novo. Animals lost the lysine biosynthesis pathway approximately 500 million years ago during early vertebrate evolution. The dinosaurs would have required dietary lysine regardless of any genetic modification. Therefore, the “contingency” provides zero additional biocontainment—it is entirely redundant.

2. Lysine is Abundant in Natural Food Sources

Based on USDA nutritional databases, lysine is widespread in both plant and animal food sources. Plant sources include legumes (soybeans, lentils, beans) containing 1-2% lysine by dry weight, seeds and grains with 0.2-0.8% lysine, and grasses and leafy vegetation with 0.3-0.6% lysine. Animal sources are even richer: insects contain approximately 2-3% lysine by dry weight, while vertebrate muscle tissue, fish, and eggs contain 1.5-2.5% lysine by weight.

Estimated lysine intake for large theropods (carnivorous dinosaurs):

Note: The following are rough extrapolations from modern vertebrate nutritional requirements and are not based on direct measurements of dinosaur metabolism. Assuming an estimated daily food intake of 50-100 kg meat (scaled from modern large carnivores) and lysine content of meat at approximately 1.5-2.0 g/100g, the estimated daily lysine intake would be 750-2000 g. Compared to an estimated lysine requirement of approximately 10-50 g/day (scaled from mammals, though highly uncertain), even conservative estimates suggest 10-100× excess lysine intake.

Estimated lysine intake for herbivorous dinosaurs:

Assuming estimated daily vegetation consumption of hundreds of kg for sauropods and lysine content in plant matter of 0.3-1.0% dry weight, the estimated daily lysine intake would be hundreds of grams. This substantially exceeds the likely requirement of 50-200 g/day when scaled from large herbivorous mammals.

Key Point: Even consuming exclusively grass, leaves, or insects would likely provide sufficient lysine to meet metabolic needs, assuming dinosaur requirements scaled similarly to modern vertebrates.

3. Timescale of Lysine Deficiency is Impractical

Lysine deficiency symptoms develop slowly: immune system impairment occurs over weeks to months, growth retardation takes months, and muscle wasting progresses over months to years. Lethality from severe deficiency requires months to years. A dinosaur escaping into the wild would eat naturally available food and immediately obtain sufficient lysine, never developing deficiency symptoms. The timescale mismatch is fatal to the strategy: containment must occur in minutes to hours (the escape window), while lysine deficiency lethality takes months to years. The result is a completely ineffective biocontainment strategy.

4. Better Biocontainment Strategies

If the goal is preventing escaped dinosaurs from surviving or reproducing, several approaches would be more effective than the lysine contingency.

Metabolic Dependencies: Creating auxotrophy for synthetic amino acids not found in nature (such as D-amino acids or unnatural amino acids requiring continuous supplementation), nucleotide auxotrophy (e.g., thymine requirement), or vitamin/cofactor dependencies (e.g., engineered B12 requirement) would provide genuine containment.

Genetic Kill Switches: Conditional lethality genes requiring antidote molecules, thermosensitive essential genes that allow survival only at controlled temperatures, or light-dependent survival mechanisms requiring specific UV or wavelength exposure offer programmed containment.

Reproductive Control: All-female populations (as attempted in Jurassic Park), meiotic drive systems ensuring sterility, or genetic incompatibility with wild relatives would prevent population establishment.

Environmental Dependencies: Temperature-sensitive phenotypes surviving only in controlled climates or organisms requiring specific atmospheric pressure or composition would restrict habitat range.

Conclusion: How This Affects My View of the Lysine Contingency

The Lysine Contingency is scientifically flawed as a biocontainment strategy and represents a misunderstanding of vertebrate nutritional biochemistry. The strategy fails on four fundamental levels: (1) it is not a contingency since lysine is already naturally essential in all vertebrates, making the modification redundant; (2) it is not limiting since lysine is abundant in nearly all natural food sources; (3) it is not fast-acting since lysine deficiency takes months to years to be lethal in large vertebrates; and (4) it provides no additional biocontainment barrier beyond natural biology.

From a biosafety perspective, the lysine contingency demonstrates the risk of “security theater” in synthetic biology—creating the appearance of control without meaningful containment. Real biocontainment requires dependencies on synthetic or artificial inputs not present in natural ecosystems. Modern synthetic biology approaches include unnatural amino acid dependencies (e.g., amber suppressor systems with synthetic tRNAs), genetic kill switches (toxin-antitoxin modules, essential gene knockout with complementation), orthogonal genetic systems (expanded genetic code, xenobiology with XNA), and metabolic dependencies on synthetic nutrients or specific light wavelengths.

Narrative function in Jurassic Park: The flawed lysine contingency serves as a plot device illustrating InGen’s overconfidence and foreshadows that all their control measures will fail (“Life finds a way”). It highlights the dangers of inadequate risk assessment and overconfidence in genetic engineering safeguards.

Lessons for modern synthetic biology: Biological containment is extremely difficult and requires multiple redundant safeguards. Single-point dependencies, especially on naturally occurring molecules, are inadequate. Rigorous testing and evolutionary escape rate measurements are essential for any containment strategy.