Week 9 HW: Cell Free Systems

Homework Part A: General and Lecturer-Specific Questions

General homework questions

Exercise 1

Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production.

Cell-free systems allow full and direct control of reaction conditions and components, enabling rapid and flexible experimentation. Here’s a table with the main advantages of cell-free vs in vivo:

Cases where cell-free is more beneficial

• Expression of toxic proteins (e.g., antimicrobial peptides)
• Incorporation of non-natural amino acids
• Expression of membrane proteins with detergents/liposomes
• Rapid prototyping of genetic circuits

Exercise 2

Main components of a cell-free expression system and their role

Exercise 3

Protein synthesis consumes large amounts of ATP and GTP. Because cell-free reactions lack the metabolic machinery of living cells, these energy molecules are rapidly depleted unless they are regenerated, which causes protein synthesis to stop and reduces yield.

A common way to maintain ATP supply is the phosphoenolpyruvate (PEP) system, in which PEP donates a phosphate group to ADP via pyruvate kinase to regenerate ATP: PEP + ADP → ATP (via pyruvate kinase). Other ATP regeneration strategies include creatine phosphate in which creatine phosphate transfers a phosphate to ADP via creatine kinase to rapidly regenerate ATP and glucose-based systems where Glucose is metabolized through enzymatic pathways to continuously produce ATP over longer reaction times.

PEP and creatine phosphate favor speed and simplicity, whereas glucose-based systems are better suited for longer and more sustainable reactions. Unless the process clearly requires extended reaction time, I would start with the PEP system because it typically delivers faster and higher ATP regeneration with a relatively simple setup.

Excercise 4: Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.

Prokaryotic vs eukaryotic cell-free systems

Prokaryotic cell-free systems such as E. coli are faster and less expensive, making them suitable for producing simple proteins that do not require complex folding or post-translational modifications, such as GFP. In contrast, eukaryotic systems are slower and more costly but are better suited for proteins that require proper folding, disulfide bond formation, or eukaryotic processing, such as human antibody fragments.

Excercise 5

To optimize membrane protein expression in a cell-free system, I would design the reaction to include a membrane-like environment during synthesis, using detergents or liposomes to maintain solubility and support proper insertion. I would also optimize reaction conditions such as magnesium concentration and temperature, and add chaperones if necessary, to reduce misfolding and improve overall yield, because membrane proteins are especially prone to misfolding and insolubility in aqueous systems.

Excercise 6: Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.

Low yield in a cell-free system can result from insufficient transcription, depletion of ATP, degradation of the expressed protein, or poor folding conditions. Troubleshooting should therefore target the limiting step directly: improve template quality if transcription is weak, reinforce energy regeneration if the reaction stalls, inhibit proteases if degradation is suspected, and optimize temperature or folding support if the protein is unstable or misfolded.

Homework question from Kate Adamala

I would design a phospholipid vesicle-based synthetic minimal cell that uses the blue-light regulator EL222 to activate expression of the tyrosinase gene melA, producing melanin as a visible record of cumulative light exposure.

Question 1

A light-exposure logging synthetic minimal cell for integration into a wearable or material patch.

a)

input:

• the synthetic cell would detect blue/visible light and respond by producing melanin
• a realistic light-sensing module is EL222, a one-component blue-light activated transcription factor from Erythrobacter litoralis that binds DNA upon illumination

output:

• gradual, visible darkening that records cumulative exposure over time.
• a realistic pigment-output gene is melA, a tyrosinase gene from Rhizobium etli that has been used to generate melanin in E. coli.

b) This function could be realized by cell-free Tx/Tl alone only partially. In bulk cell-free solution, the circuit could still produce melanin, but without encapsulation it would not behave as a discrete synthetic minimal cell and would be harder to localize, stabilize, or integrate into a material as a spatially resolved light-logging unit.

c) This function could also be realized by a genetically modified natural cell. For example, E. coli can be engineered to express melA and produce melanin. A synthetic minimal cell is preferable if the goal is a compartmentalized, material-compatible system rather than a living replicating microbe.

d) The desired outcome is that the synthetic cell becomes darker as cumulative light exposure increases. In a material, a population of these vesicles would function as a distributed exposure log: more illuminated regions would accumulate more melanin and therefore appear darker than shaded regions.

Question 2

a) The membrane would be a phospholipid vesicle, for example POPC + cholesterol, because that is a standard stable composition for synthetic cell vesicles and is also used in related artificial-cell communication systems.

b) Inside the vesicle, I would encapsulate an E. coli cell-free transcription/translation system, amino acids, NTPs, salts, and cofactors, an ATP regeneration system, such as PEP + pyruvate kinase, L-tyrosine as the melanin precursor Cu²⁺ as a cofactor for tyrosinase, DNA encoding the light-response module and melanin-output module.

c) For the Tx/Tl source, a bacterial system is sufficient. The core regulator, EL222, is bacterial, and the output enzyme MelA tyrosinase does not require mammalian-specific post-translational processing to function as a pigment-producing enzyme.

d) The synthetic cell would communicate with the environment mainly through light, which crosses the membrane directly, so no membrane channel is required for the input. To simplify the system, I would preload tyrosine and copper inside the vesicle. If I later wanted continuous substrate exchange from the outside, I could add a pore such as α-hemolysin (Hla), which is commonly used in synthetic-cell communication designs.

Exercise 3 - Experimental details

a)

Lipids: POPC, cholesterol Genes: EL222 from Erythrobacter litoralis as the light-activated transcription factor; melA from Rhizobium etli as the tyrosinase gene for melanin production optional: hla for α-hemolysin if external substrate exchange is needed; Encapsulated reagents: E. coli cell-free lysate or PURE-like system, amino acids, NTPs, PEP, pyruvate kinase, tyrosine, Cu²⁺

b)

I would measure the function of the system by tracking darkening over time, using image analysis and bulk absorbance measurements. The most direct readout is the increase in visible pigmentation of illuminated vesicles relative to dark controls; microscopy could also be used to compare spatial patterns of melanin accumulation across the material.

Homework question from Peter Nguyen

Application field: Textiles / Fashion

One-sentence pitch A textile integrated with freeze-dried cell-free melanin-producing modules that develops gradual, skin-adjacent tonal changes in response to light exposure, turning the garment into an exposure-recording surface.

How it works The material would incorporate localized freeze-dried cell-free reaction zones containing the genetic and enzymatic components needed for melanin production, for example a light-responsive regulator such as EL222 coupled to a melanin-producing gene such as melA. When the textile is activated by hydration, these embedded reaction zones become functional and begin responding to light exposure by expressing tyrosinase and generating melanin from preloaded substrate. Over time, more exposed regions of the garment darken more than shaded or covered regions, creating gradients or “tan-line-like” traces directly in the material. Functionally, the textile behaves less like a conventional dyed fabric and more like a programmable, exposure-sensitive biological film.

Societal challenge or market need This concept addresses the growing interest in responsive and personalized materials in fashion and design, especially materials that are not just decorative but capable of recording use, environment, or time. It also responds to demand for alternatives to static coloration and conventional dyeing by proposing a material whose visual output is generated biologically in place. Beyond fashion, the same platform could be relevant to design objects or artistic textiles that visibly register environmental exposure.

How to address limitations of cell-free reactions

• Because freeze-dried cell-free systems require water for activation and are typically limited in duration, I would treat the material as an on-demand activation platform rather than a permanently active textile. The garment could be hydrated only when the user wants to generate a pattern or record a specific exposure event, which also helps manage stability and one-time use.
• To improve shelf life, the cell-free modules would remain freeze-dried until use and be stored in sealed conditions;
• To improve localization and handling, they could be embedded in discrete patches, printed zones, or replaceable inserts rather than distributed uniformly across the whole textile. This makes the limitation part of the design logic: the material is activated intentionally, records one event or interval, and then remains as the final artifact.

Background information (max 100 words) Space radiation can damage DNA and reduce the reliability of biological systems used for diagnostics, manufacturing, and environmental sensing during long-duration missions. This is significant because future crews will likely depend on compact biotechnology tools rather than constant resupply from Earth. It is relevant for space exploration because cell-free systems are lightweight, storable, and already attractive for use in resource-limited environments. It is scientifically interesting because it links a basic biological question - how nucleic acid damage affects gene expression - to an applied engineering problem: how to maintain functional biotechnology in space.

Molecular or genetic target (max 30 words) Integrity and expression efficiency of a PCR-amplified sfGFP DNA template after radiation-mimicking UV exposure.

How the target relates to the challenge (max 100 words) The sfGFP DNA template serves as a simple reporter for whether a biologically useful DNA sequence remains functional after damage. If radiation-like exposure degrades the template, BioBits cell-free protein expression should produce less GFP signal. This makes the target directly relevant to the space biology challenge, because many space biotechnology applications depend on DNA templates remaining intact enough to be transcribed and translated. Measuring GFP output therefore provides a practical way to estimate how radiation damage could impair future cell-free diagnostics or production systems used in spacecraft or habitats.

Hypothesis or research goal (max 150 words) My hypothesis is that increasing UV exposure, used here as a classroom-accessible proxy for radiation-induced nucleic acid damage, will reduce the ability of a PCR-amplified sfGFP DNA template to produce GFP in the BioBits cell-free expression system. I further expect that templates protected by a shielding condition, such as melanin-containing film or another UV-blocking barrier, will retain more expression than unprotected templates exposed to the same dose. The reasoning is that DNA damage should interfere with transcription and translation by reducing template integrity, while a protective barrier should lower that damage. The research goal is to test whether cell-free fluorescence output can function as a simple readout of DNA stability under space-relevant stress and whether a lightweight protective strategy improves performance.

Homework question from Ally Huang

Background information: Space radiation can damage DNA and reduce the reliability of biological systems used for diagnostics, manufacturing, and environmental sensing during long-duration missions. This is significant because future crews will likely depend on compact biotechnology tools rather than constant resupply from Earth. It is relevant for space exploration because cell-free systems are lightweight, storable, and already attractive for use in resource-limited environments. It is scientifically interesting because it links a basic biological question: how nucleic acid damage affects gene expression to an applied engineering problem: how to maintain functional biotechnology in space.

Molecular or genetic target: Integrity and expression efficiency of a PCR-amplified sfGFP DNA template after radiation-mimicking UV exposure.

How the target relates to the challenge: The sfGFP DNA template serves as a simple reporter for whether a biologically useful DNA sequence remains functional after damage. If radiation-like exposure degrades the template, BioBits cell-free protein expression should produce less GFP signal. This makes the target directly relevant to the space biology challenge, because many space biotechnology applications depend on DNA templates remaining intact enough to be transcribed and translated. Measuring GFP output therefore provides a practical way to estimate how radiation damage could impair future cell-free diagnostics or production systems used in spacecraft or habitats.

Hypothesis or research goal: My hypothesis is that increasing UV exposure, used here as a classroom-accessible proxy for radiation-induced nucleic acid damage, will reduce the ability of a PCR-amplified sfGFP DNA template to produce GFP in the BioBits cell-free expression system. I further expect that templates protected by a shielding condition, such as melanin-containing film or another UV-blocking barrier, will retain more expression than unprotected templates exposed to the same dose. The reasoning is that DNA damage should interfere with transcription and translation by reducing template integrity, while a protective barrier should lower that damage. The research goal is to test whether cell-free fluorescence output can function as a simple readout of DNA stability under space-relevant stress and whether a lightweight protective strategy improves performance.

Experimental plan:

• I will amplify an sfGFP template with the miniPCR and divide it into groups:
  • no UV exposure
  • low UV
  • high UV, and
  • UV plus shielding
• After treatment, each sample will be added to BioBits cell-free reactions. Negative controls will include reactions with no DNA template; positive controls will include unexposed template.
• GFP fluorescence will be measured with the P51 Molecular Fluorescence Viewer and quantified by image intensity or relative brightness. The main data will be fluorescence level across conditions, which will indicate how template damage affects expression and whether the shielding condition preserves function.

Homework Part B: Individual Final Project

general info / link for my slide in the CT slide deck

Here’s my slide in the CT slide deck

Title: Engineering Tunable Skin Pigment Expression in Engineered Living Materials

Aim 1: Generate base data on melanogenesis by mapping key pathways and build an initial genetic circuit informed by this base data to produce tunable pigmentation (eumelanin-biased outputs for darker tones and pheomelanin-biased outputs for warmer tones).

Aim 2: Expand and refine the circuit aiming for selecting envisioned great candidates for wet-lab experimentation. Experiments planning.

Aim 3: Empirical essays to explore how variables such as pigment amount, distribution, and system conditions affect the final material output. Companies: BioFabricate; Cultivarium

Industry Council Companies: BioFabricate and Cultivarium I selected them because they each address a different core part of my project: Biofabricate could potentially bring a strong expertise on how to translate embedding melanin-related genetic circuits into a desirable (aesthetic and functional) engineered living material, while Cultivarium is well aligned with the wet-lab side of the project, particularly chassis selection, non-model organism engineering, and the practical challenge of implementing and optimizing the circuit in a host such as Komagataeibacter rhaeticus.

Submit the Final Project selection form.

Started planning how I will write my final project documentation based on the guidelines

To be done by April 10 at 11PM ET. Prepare your first DNA order and put it in the “Twist (MIT)” or “Twist (Nodes)” tab of the 2026 HTGAA Ordering: DNA, Reagents, Consumables spreadsheet, as appropriate.