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?

   New Types of Products to Create

   Synthetic microbial communities:

   Engineered ecosystems for environmental remediation, agriculture, or even space colonization (stable, self-repairing, self-adapting).

   Rather than engineering just individual organisms, we could design whole communities of organisms — bacteria, fungi, plants, even synthetic cells — that work together in a stable, self-adapting way to solve a problem.

   • Bioremediation:
     • A designed microbial community that degrades oil spills, absorbs heavy metals, fixes nitrogen, and restores soil health — all at once.
   • Agriculture:
     • A root microbiome that feeds crops, protects them from pests, adjusts to drought, and improves yield without fertilizers.

   Enzymes that break down plastics, toxins, or pollutants efficiently under diverse conditions.

   Many synthetic chemicals (plastics, pesticides, industrial waste) persist in the environment because natural enzymes evolved before these pollutants existed — they aren't designed to break them down.

   Challenges today:

   • Finding or evolving the right enzymes takes years of slow, trial-and-error laboratory evolution.
   • Enzymes often work only under lab conditions, not in harsh environmental settings (e.g., salty oceans, acidic mines).

   How full automation would help:

   • Screen millions of enzyme variants in parallel, testing them under dozens of environmental stresses.
   • Directed evolution at massive scale: Faster cycles of mutation and selection could lead to super-enzymes.
   • Synthetic metagenomics:
     • Rebuild entire communities with optimized enzymatic networks, not just one enzyme at a time.

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

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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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?

   Pigment	Genes Needed	Explanation
   Lycopene	crtE, crtB, and crtI	These three genes, (often from Erwinia herbicola), encode enzymes that convert farnesyl diphosphate (FPP) into lycopene .
   Beta-carotene	crtE, crtB, crtI, and crtY	Beta-carotene production requires the same lycopene pathway genes plus crtY, which encodes a lycopene cyclase that converts lycopene into beta-carotene .

   

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

   Purpose	Explanation
   Selection marker	After transformation, not all E. coli cells will successfully take up the plasmid. By adding an antibiotic (like chloramphenicol) to the growth media, only the cells that have the plasmid (and thus the resistance gene) will survive and grow .
   Maintaining plasmid presence	During cell division, bacteria could lose plasmids. The antibiotic pressure ensures that cells keep the plasmid, because losing it would make them sensitive and they would die. This maintains stable expression of the desired genes.

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

   • Richer media + fructose + 30°C = best for high pigment production.
   • Poorer media + no fructose + 37°C = faster cell growth but possibly lower pigment yields.

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

   Aspect	Description
   What OD600 measures	OD600 (Optical Density at 600 nm) measures how much light is scattered by a bacterial culture. It gives an estimate of cell density — the more cells, the more light is scattered.
   Why 600 nm?	600 nm is a wavelength where bacterial cells scatter light well without strong absorption by pigments or media components .

   • High OD600 = lots of bacterial growth.
   • Low OD600 = little bacterial growth.
   • To compare pigment production, it is needed to normalize pigment absorbance to OD600 so measuring is pigment per cell, not just total color.

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?

   Experimental Setup	Why Acetone is Useful
   Pigment extraction (e.g., chlorophyll, carotenoids)	Acetone breaks cell membranes and solubilizes pigments while precipitating proteins and cell debris, making pigment measurement easier.
   Lipid extraction (part of some protocols like Bligh-Dyer method)	Acetone can help in lipid extraction by dissolving fats and precipitating non-lipid material.
   Metabolite extraction (e.g., small hydrophobic molecules)	Acetone can release and concentrate hydrophobic metabolites while removing proteins and insoluble debris.
   Protein precipitation before proteomic analysis	Acetone precipitates proteins for later resuspension and analysis, such as before mass spectrometry.
   Sample cleanup in chromatography (e.g., HPLC sample prep)	Acetone can be used to dehydrate or clean up biological samples before loading into a chromatography system.

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

   Reason	Explanation
   Ease of cultivation	E. coli grows much faster and more predictably than Erwinia herbicola. It is easier to scale up for industrial production.
   Genetic tools available	E. coli has well-developed genetic engineering tools (plasmids, promoters, gene editing), allowing precise control of pigment production .
   Safety and regulatory familiarity	E. coli (especially laboratory strains) are considered safe (biosafety level 1) and are widely accepted for production of food additives and pharmaceuticals.
   Higher yields possible	Through metabolic engineering, E. coli can be optimized to produce much higher amounts of lycopene and beta-carotene than Erwinia naturally would .
   Simplified downstream processing	Harvesting pigments from E. coli can be easier and cleaner than from Erwinia, reducing production costs.

Optional Learning Module

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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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