Homework Part A

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?

   Questions

   • Can we predict and direct cellular reprogramming outcomes with high precision?
   • How do non-coding RNAs coordinate large-scale physiological responses?
   • What role do RNA modifications play in cell fate determination at the single-cell level?

   New types of products

   • Personalized and adaptive gapmer-based therapeutics.
   • Real-time biosensing systems for continuous health monitoring.
   • Modular platforms for rapid prototyping of vaccines or gene therapies.

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

   If I could manufacture metric tons of any protein, I would produce insulin, despite biotechnological advances, insulin remains inaccessible to many people around the world. Producing it on a large scale could drastically reduce its cost and ensure global access for diabetes sufferers.

Homework Part B

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

   • For lycopene:

     • crtE: Geranylgeranyl diphosphate synthase.
     • crtB: Phytoene synthase.
     • crtI: Phytoene desaturase.

     The introduction of the crtE, crtB and crtI genes from Pantoea agglomerans into E. coli results in increased lycopene production compared to homologous genes from other species, as it aids in increased catalysis in the conservation of isoprenoid precursors of lycopene.

   • For beta-carotene:

     • crtY: Licopeno β-ciclasa.

     This gene catalyzes the cyclization of lycopene to form β-carotene. Coexpression of crtE, crtB, crtI and crtY in E. coli has enabled efficient production of β-carotene .

   

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

   Plasmids transferred into Escherichia coli typically contain an antibiotic resistance gene as a selectable marker, which allows researchers to identify and maintain only the bacterial cells that have successfully taken up the plasmid.

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

   Can significantly influence the growth rate of E. coli and the expression level of recombinant products such as carotenoids

   • Culture media:
     • Rich media like LB support higher growth but can introduce variability.
     • Defined media provide better control and reproducibility.
   • Fructose vs. glucose:
     • It may affect catabolite repression and change flux through the MEP or mevalonate pathways.
     • Some studies report increased carotenoid yield with sugars like fructose, compared to glucose.
   • Temperature:
     • Lower temperatures
       • Slower bacterial growth but may improve protein folding and enzyme stability, increasing final carotenoid yield.
     • Higher temperatures
       • Rapid growth, but risk of metabolic burden, protein misfolding, and stress response, which may reduce product yield.

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

   OD₆₀₀ (Optical Density at 600 nm) is a spectrophotometric measurement used to estimate the cell density.

   • Monitor growth over time, especially during overnight culture.
   • Compare growth rates under different conditions (media, temperature, fructose).
   • Normalize carotenoid yields, to ensure that differences in pigment production are not just due to differences in biomass.

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

   Acetone is an organic solvent that is used in many experimental protocols to precipitate cellular components.

   • Extraction of pigments, for example chlorophyll from plants or algae.
   • Lipid extraction from microbial or plant cells.
   • Protein precipitation for proteomics.
   • Sample preparation for HPLC, spectrophotometry, or TLC when the target compound is soluble in organic solvents.

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

   • Cost-effective and high-performance production:
     • It is easy to grow on inexpensive media and is a very versatile expression system for large-scale production.
   • Genetic Control and Customization:
     • Optimize metabolic pathways to produce higher yields of lycopene and β-carotene.
   • Scalability for Industrial Applications:
     • It facilitates its use for the manufacture of commercial products.
   • Avoiding Contaminants and Toxins:
     • Minimizes contamination risks and ensures a safer final product.