Homework Part A

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> These homework questions are based on lecture questions! Mandatory for Committed Listeners.

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Patrick Boyle’s Lecture Questions:

1. Assume that all of the molecular biology work you'd like to do could be automated, what sort of new biological questions would you ask, or what new types of products would you make?

   Ans: If molecular biology workflows were fully automated, researchers could pursue high-throughput synthetic biology to explore biological systems at unprecedented scales. For example:

   1. Combinatorial genetics: Systematically testing millions of gene circuit variants to map epistatic interactions in metabolic pathways or signaling networks
   • Evolutionary prototyping: Running continuous directed evolution experiments with AI-guided mutagenesis to engineer proteins with novel functions (e.g., plastic-degrading enzymes)
   • Personalized gene therapies: Rapidly manufacturing patient-specific viral vectors or mRNA vaccines tailored to individual biomarker

   Automation would also enable industrial-scale biological manufacturing of:

   • Programmable biomaterials: Self-assembling protein nanostructures for targeted drug delivery or eco-friendly textiles
   • Living sensors: Engineered organisms for real-time environmental monitoring of toxins/pathogens

2. If you could make metric tons of any protein, what would you make and what positive impact could you have?

   Ans: With metric-ton protein production capabilities:

   Protein	Impact
   CRISPR-Cas nucleases	Enable low-cost gene editing therapies for genetic diseases (e.g., sickle cell anemia)
   Nitrogenase	Reduce synthetic fertilizer use by engineering crops to fix atmospheric nitrogen directly
   CO₂-fixing enzymes	Scale carbon sequestration via synthetic metabolic pathways in industrial bioreactors
   Broad-spectrum antivirals	Deploy affordable prophylactic proteins against pandemic viruses in developing nations

   Most transformative: Industrial-scale production of cell-free protein synthesis systems themselves, enabling decentralized biomanufacturing in resource-limited settings. This could democratize access to vaccines and therapeutics while reducing cold-chain dependencies

Homework Part B

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> These homework questions are based on the Bio Production Lab! Mandatory for both Committed Listeners and MIT/Harvard students.

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<aside> <img src="/icons/push-pin_green.svg" alt="/icons/push-pin_green.svg" width="40px" /> Key Links: http://docs.google.com/document/d/15-tlrejgbbr4FMpA6rKogTjlv6qXJhFqQm7o_Ppfh-I/edit?tab=t.0#heading=h.jyt74412izch

Key Papers:

1. Gene expression pattern analysis of a recombinant Escherichia coli strain possessing high growth and lycopene production capability when using fructose as carbon source

2. Improvement of Biomass Yield and Recombinant Gene Expression in Escherichia coli by Using Fructose as the Primary Carbon Source </aside>

3. Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?

   Ans:

   • Lycopene: Transfer of crtE (GGPP synthase), crtB (phytoene synthase), and crtI (phytoene desaturase) from Erwinia herbicola enables lycopene biosynthesis in E. coli via the MEP pathway
   • Beta-carotene: Requires the additional crtY (lycopene β-cyclase) gene to convert lycopene into β-carotene

4. Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?

   Ans: Plasmids (e.g., pAC-LYC/pAC-BETA) include chloramphenicol resistance to select for E. coli successfully incorporating the plasmid. Only cells with the plasmid survive in antibiotic-supplemented media, ensuring culture purity

5. What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?

   Ans:

   • Media (LB vs. 2YT): Richer media (2YT) increases biomass and pigment yield due to enhanced nutrient availability
   • Fructose: Reduces acetate accumulation, improving cell health and recombinant protein yield
   • Temperature: Lower temps (30°C) may reduce metabolic stress, enhancing pigment stability; 37°C accelerates growth but risks plasmid loss

6. Generally describe what “OD600” measures and how it can be interpreted in this experiment.

   Ans: OD600 measures the optical density at 600 nm which correlates with cell density via light scattering by bacterial cells. This normalizes pigment absorbance (474 nm/456 nm) to cell concentration, enabling comparison of production efficiency across samples.

7. What are other experimental setups where we may be able to use acetone to separate cellular matter from a compound we intend to measure?

   Ans: Acetone precipitates cellular debris while solubilizing hydrophobic compounds (e.g., carotenoids) so it can be used for DNA or Protein extraction.

8. Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?

   Ans:

   • Faster growth: E.coli reaches higher densities quicker, enabling scalable bioproduction
   • Genetic tractability: Easier to engineer pathways (e.g., MEP optimization) for higher yields
   • Reduced complexity: Avoids native regulatory hurdles in Erwinia and simplifies industrial fermentation

   Example: E.coli MG1655 produces 1,595 mg/L lycopene in optimized media, outperforming native producers

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Learning Module

<aside> <img src="/icons/exclamation-mark_orange.svg" alt="/icons/exclamation-mark_orange.svg" width="40px" /> These homework questions are based on the Bio Production Lab! Optional but encouraged! These questions are designed to help you start thinking more closely about DNA Design.

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Exploring Carotenoid Bioproduction

1. Getting in Touch with the Carotenoid Metabolic Pathway

1. Identify Key Enzymes
   • In the carotenoid biosynthesis pathway (for carotene and lycopene), what are the principal enzymes involved? List them in order.
2. Rate-Determining Step
   • Within this pathway, which biochemical reaction is generally the slowest and thus “rate-limiting”?
   • Which enzyme catalyzes this rate-determining step?

2. Designing a DNA Construct for Carotene Production

You will design a DNA construct aimed at producing carotenoids (e.g., lycopene or beta-carotene) in either E. coli or S. cerevisiae. Work through the questions in sequence to build a conceptual and practical understanding of how to engineer a biosynthetic pathway.