Week 1 HW: Principles and Practices

Question 1

In the 1980’s, Keith Wood became the first to make a tobacco plant glow using firefly luciferase. However, he faced a critical limitation: they could not synthesize their own luciferin, requiring an external luciferin spray to emit light. ¹ As Wood pivoted to fungal pathways, the firefly route has been largely abandoned despite its superior light efficiency. However, recent breakthrough have reopened this door, including the discovery of spontaneous benzoquinone + cysteine L-luciferin formation, and the identification of ACOT1 in D-luciferin transformation. My project aims to put these breakthroughs together, engineering plants capable of bioluminscence through firefly pathways.

As we know:

• Fireflies create an enzyme called luciferace that can oxidize D-luciferin.
• Luciferase can be put synthetically engineered into plants, but must be sprayed with luciferin to synthesize luciferin.
• Kanie et al. (2016) discovered arbutin can spontaneously react with L-cysteine (naturally found in plants) to create L-luciferin.
• Zhang (2020) discovered ACOT1 as the enzyme responsible for catalyzing the reaction from L-luciferin to D-luciferin.
• In theory, these reactions could make a plant glow with many foreseen and unforeseen modifications.

My proposed reaction:

Arbutin = 
GLUCOSE + HYDROQUINONE 

Glucose -0 -Hydroquinone gets cut with BGL (β-glucosidas).

Hydroquinone -> 
Benzoquinone

We inject our first gene (lactase) to remove two -0H groups, creating benzoquinone. Important to make sure the benzoquinone is trapped where it meets cysteine immediately or its proteins can damage the cytoplasm of the plant

Benzoquinone + Cysteine
= 
L-LUCIFERINE

As we have discovered, benzoquinone spontaneously reacts with cysteine under certain conditions, producing L-Luciferin

L-Luciferin + ACOT1 = 
D-luciferin AND L-LUCIFERIN

We inject ACOT1 (naturally occurring in the firefly) to catalyze the reaction into D-luciferin (the form that glows)

D-luciferin + ATP = LUCIFERYL-ADENYLATE + PPI

LUCIFERYL-ADENYLATE + 02 = OXYLUCIFERIN + CO2 + AMP 

OXYLUCIFERIN -> 
OXYLUCIFERIN + PHOTON

(Well understood steps of how d-Lucefirin is created into light)

Plant already supplies: Arbutin, Cysteine, ATP, 02

We add 4 enzymes: BGL, Laccase, ACOT1, Luciferase

Question 2

Beyond the ethical application of synthetic genomics, an important goal would be to ensure the plant will not be detrimental or disrupt natural ecosystems. (Example: GloFish goldfish being an invasive species after someone released them in Brazil.) Goals pertaining to this specific issue will be broken down below.

Option 1: Lab-work will be contained and carefully scrutinized to ensure plants are not accidentally released. This includes but is not limited to: proper disposal, proper containment, proper sanitization.

Option 2: Plants will eventually be tested in greenhouse settings, or other settings replicating natural ecosystems to verify no immediate or obvious harm and gather data in case of contamination.

Option 3: Plants that are modified will be non-invasive and common (ex. Petunias), and will be modified only to the extent needed for autonomous self-bioluminescence.

Option 4: Research in later stages will be done with the aim of open-environment release, where doing so will be safe for natural ecosystems.

• ME = Most Effective
• MOD = Moderately Effective
• RE = Relatively Effective
• SE = Somewhat Effective
• MIN = Minimally Effective
• n/a = n/a

A second goal is to remain responsible with tools and research. As a newcomer to synthetic biology, I want to ensure proper attribution to the work of the reseachers who make this possible.

Option 1: Prevent misuse of plasmids/enzymes/resources from previous academic papers.

Option 2: Be respectful to my lab space and peers, maintaining proper safety and etiquette.

• ME = Most Effective
• MOD = Moderately Effective
• RE = Relatively Effective
• SE = Somewhat Effective
• MIN = Minimally Effective
• n/a = n/a

Question 3

Action 1

Purpose: As this is such a new field, these is little regulation on the genetic modification of plants for commercial/art purposes. I propose a more thorough, environmental review before the release of plants.

Design: Researchers would need to submit their own opinions and data, while federal regulators would need to propose their own processes.

Assumptions: That everyone will be acting in good faith, not unnecessarily over or under regulating, and that regulators have the knowledge required to make educated decisions.

Risks: Over-regulation would cause extreme bureaucracy in the the bio-plant space, and cause resentment in the scientific community. Under-regulation might cause detrimental harm to natural ecosystems in Canada.

Action 2

Purpose: People lack incentive or the freedom to purse such projects. Companies can make it more attractive for workers to purse bioengineering for commercial or art purposes.

Design: Corporations or the government of Canada can create programs that incentivize bio-plant discoveries, making it more accessible and attractive for individuals.

Assumptions: Corporations or government programs would be executed well-enough for progress, and enough attention would be directed at students/workers to ensure some level of success.

Risks: Corporations may over-rely on potential profits, pushing individuals into projects that may not be beneficial for society. This is where Action 1 regulations should ideally be applicable. Programs may not be executed in good faith or passion, leaving people uninspired and destitute.

Action 3

Purpose: People lack access to labs, and most places do not have community bio-labs. Autonomous labs will still take time to be implemented on a wider scale. People should be able to get certificates for level-1 biohazard for at-home labs from the government.

Design: A certain certification process should undergo with organizations representing the government. Once you prove you can meet certain standards, a visit should be granted to confirm. If granted, you are certified for a certain amount of time until you re-certify.

Assumptions: Individuals engaging in home labs are responsible, and strive to truly meet these standards at all time. Federal regulators act in good faith, and are responsible with their certifications.

Risks: Individuals running home labs accidentally contaminate or cause injury to themselves or others.

Week 2 Lecture Prep

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?

Error rate of polymerase in nature is 1 x 10−6 or 1:106

Human genome: 3.2 Gbp (billion base pairs) or 3.2×109 bases

So if error rate is 1 × 10−6 per base: (1×10−6)×(3.2×109) = 3.2×103 = 3,200 errors

Biology deals with this through the MutS Repair System, where a DNA repair protein scans the replicated DNA, and binds where there are errors in the pairing. Once the MutS is binded, that part of the strand is cut, and the exonuclease removes the error. Lastly, the DNA polymerase resynthesizes the correct pairing, ligase seals it, and the error is removed. This system improves fidelity by 100-1000x, effectively lowering the error number in human genomes to 0.3-1 per copy.

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?

The average human protein is estimated to be around 400 amino acids ( 1036 bp). There are an average 3 numbers of different codons that encode the same amino acid, therefore the number of possible sequencing is ≈3400, which is an absurdly high number. However, this does not translate in practice. Firstly, different organisms prefer different codons, which is why we optimize our codons depending on our project. Biology still cares about the nucleotide sequence, despite identical codon sequences. Other reasons include the mRNA behaving differently depending on the structure, certain codes negatively affecting how the protein folds, RNA instability, among other practical hurdles. Slide 62 has an interesting argument as to why optimal alphabet size falls in the tens, not hundreds.

Homework Questions from Dr. LeProust

What’s the most commonly used method for oligo synthesis currently?

The most commonly used method is phosphoramidite solid-phase DNA synthesis, of which Twist is the main commercial provider for. While phosphoramidite has been optimized since its founding in 1981, the overall method has remained the same. The DNA is built one base at a time using a repeating cycle. First a DNA strand is attached to a solid surface, and a DMT protecting group blocks the 5’ end. Once a chemical acid removes the DMT and exposes the 5’OH, after which a phosphoramidite nucleotide is added to the free 5’-OH (~99–99.5% per cycle efficiency). Since not every strand couples successfully, Twist chemically caps the failed strand. Then the new linkage is chemically oxidized to form a stable bond. Then the cycle repeats.

Why is it difficult to make oligos longer than 200nt via direct synthesis?

Because with every additional nucleotide, the yield drops since efficiency is not at 100%, but 99.5%. Therefore, for an 100 nucleotide length strand, 0.995100 ≈ 60%, where only 60% of molecules will reach their full length, while the rest are truncated somewhere else.

Why can’t you make a 2000bp gene via direct oligo synthesis?

Because at 2000bp, the percentage of successful molecules will essentially be at 0. (0.9952000 ≈ 0.005%) There would be almost no full length molecules. This is a hard, biological wall we are yet to solve.

Homework Question from George Church

Choose ONE of the following three questions to answer; and please cite AI prompts or paper citations used, if any. 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 in animals are Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine. These are obtained through an animal’s diet, considered essential as they generally cannot be synthesized by animals. The “lysine contingency” is an idea that was popularized by the franchise Jurassic Park. In the movies, dinosaurs were engineered to be unable to make the lysine amino acid without a human-supply, so that in theory, they would not be able to survive without human guidance. This was done to prevent escape, and safety regulations. However, this wouldn’t seem to work in real life. Animals are already “lysine contingent”; we are unable to synthesize lysine. That’s why lysine is found in a wide variety of animal diets, and can be easily supplemented by food. Virtually all food sources and ecosystems contain lysine, scavenging would already easily supply it. Furthermore, there are many other amino acids we require. Signalling one out is arbitrary. Effective containment would rely on dependencies that are rare in nature.

Other References

Iwano, S., Sugiyama, M., Hama, H., Watakabe, A., Hasegawa, N., Kuchimaru, T., … Miyawaki, A. (2018). Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science, 359(6378), 935–939. https://doi.org/10.1126/science.aaq1067

Kanie, S., Abe, K., Hirano, T., & Niwa, H. (2016). One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine. Scientific Reports, 6, 24794. https://doi.org/10.1038/srep24794

Zhang, R., Chen, L., Jiao, J., Zhou, Y., Liu, Y., & Wang, Y. (2020). Genomic and experimental data provide new insights into luciferin biosynthesis and bioluminescence evolution in fireflies. Scientific Reports, 10, 15882. https://doi.org/10.1038/s41598-020-72900-z

Applicable AI Promots: Summarize Keith Wood’s lifework and research in firefly related plant bioluminescence.

(Image of example table) explain what they mean by options, and how the graph works.

Explain the MutS mismatch repair system in detail.

How many different ways are there to code (DNA nucleotide code) for an average human protein? Why doesn’t it translate in practice?

Explain how phosphoramidite solid-phase DNA synthesis works in detail.

Is lysine in all of our foods?

Google What are the 10 essential amino acids in all animals (https://www.sciencedirect.com/science/article/pii/S216183132201273X)

What is the “lysine contingency” (https://jurassicpark.fandom.com/wiki/Lysine_contingency#:~:text=The%20Lysine%20Contingency%20was%20a,acquire%20Lysine%20by%20eating%20herbivores.)