Week 9 HW: Cell-Free Systems

Part A: General and Lecturer-Specific Questions

Advantages of cell-free protein synthesis (CFPS) over in vivo expression

1. Greater experimental control
2. Open reaction environment
3. Faster design-build-test cycles
4. Toxic protein production
5. Better access to non-natural chemistry
6. Easier monitoring and automation

Cases where cell-free expression is more beneficial than cell production

Case 1: Toxic proteins Example:

• pore-forming membrane proteins
• antimicrobial peptides
• viral toxins

In vivo expression may kill the host, reduce growth, and cause plasmid instability.

Case 2: Rapid prototyping of genetic circuits CFPS allows same-day testing without cloning into cells:

• promoters
• ribosome binding sites
• regulatory networks

Main components of a cell-free expression system

1. Cell lysate (extract): ribosomes, tRNAs, translation factors, aminoacyl-tRNA synthetases, metabolic enzymes
2. DNA or mRNA template: promoter, ribosome binding site, coding sequence, terminator
3. Amino acids
4. Energy source: phosphoenolpyruvate (PEP), creatine phosphate, glucose, maltodextrin
5. Nucleotides
6. Salts and cofactors
7. Optional additives: chaperones, detergents, liposomes, disulfide bond catalysts, protease inhibitors

Why energy regeneration is critical

Protein synthesis is extremely energy intensive. Each peptide bond formation requires ATP, GTP, tRNA charging, and ribosome translocation. Energy sources are rapidly depleted during protein synthesis, and accumulation of inorganic phosphate due to their cleavage additionally inhibits reactions.

Example method for continuous ATP supply Phosphoenolpyruvate (PEP) regeneration system

PEP + ADP → Pyruvate + ATP (requires pyruvate kinase)

This is a simple method for fast ATP regeneration commonly used in CFPS. Although it is relatively expensive and does not solve phosphate accumulation-induced inhibition of synthesis.

Prokaryotic vs. eukaryotic cell-free systems

Prokaryotic CFPS are ideal for proteins with simple folding and no glycosylation needed, which also express highly in E. coli: e.g. GFP. In this case, there are no obstacles for choosing this inexpensive and fast method.

Human monoclonal antibodies, such as IgG, require disulfide bonds, glycosylation and complex folding. Eukaryotic CFPS provide ER-like folding conditions, post-translational modifications and better assembly.

Designing a CFPS experiment for membrane protein expression

Membrane proteins are difficult because they tend to aggregate, misfold, and precipitate outside membranes. Also, they contain hydrophobic domains.

1. Choose suitable CFPS system: E. coli lysate with nanodiscs or eukaryotic lysate with microsomes
2. Add membrane mimetics: detergents / liposomes / bicelles / nanodiscs
3. Optimize lipid composition: proportions of phosphatidylcholine + phosphatidylglycerol + cholesterol
4. Include chaperones
5. Control translation rate: lower temperature, reduced magnesium, weaker promoters (?)

Low protein yield: reasons and troubleshooting

HW question from Kate Adamala: Example Synthetic Minimal Cell Design

1. a) Function: Smart inflammation-sensing therapeutic synthetic cell This synthetic minimal cell is designed to detect inflammatory signals associated with intestinal disease (such as inflammatory bowel disease, IBD) and respond by producing an anti-inflammatory therapeutic protein. It would:

• Sense inflammatory biomarkers in the gut
• Process the signal internally
• Produce and release an anti-inflammatory molecule only when inflammation is detected

Input

• nitric oxide (NO)
• reactive oxygen species (ROS)
• inflammatory cytokines (e.g. TNF-α)

Output

• interleukin-10 (IL-10), an anti-inflammatory cytokine or
• a fluorescent reporter (GFP) for testing purposes

Inflammation (NO) → activation of promoter → IL-10 production → therapeutic effect

1. b) Encapsulation over CFPS A pure bulk cell-free system could detect NO and express IL-10 transiently. However, encapsulation is highly advantageous because it provides:

• compartmentalization
• protection from degradation
• localized delivery
• controlled diffusion
• sustained operation

1. c) Could this function be realized by genetically modified natural cells? Yes.

But, synthetic minimal cells offer advantages:

• no replication
• reduced biosafety risks
• no mutation/evolution
• easier containment
• programmable lifespan
• lower immune complexity

1. d) Desired outcome Minimal synthetic cell for proposed purpose should:

• remain inactive under healthy conditions
• detect inflammation-associated NO
• produce IL-10 locally
• reduce inflammation without systemic immunosuppression
• degrade safely after use

2. a) Membrane composition POPC (phosphatidylcholine) + cholesterol. Also, DOPE and cardiolipin if there would be resources for these details.

3. b) Encapsulated contents

• Ribosomes, tRNAs, amino acids, ATP/GTP, RNA polymerase, translation factors
• DNA encoding NO-responsive transcription factor system, IL-10 therapeutic output, GFP (or other) reporter
• Phosphoenolpyruvate (PEP), pyruvate kinase, or glucose metabolism enzymes
• GroEL/ES or DnaK, glutathione, DTT

2. c) Choice of Tx/Tl system

• Prokaryotic as proof-of-concept
• Mammalian or hybrid systems may be preferable for therapeutic-grade IL-10: it could be syntheside without mammalian post-translational modifications but it is probably unavoidable for immune acceptance.

2. d) Communication with the environment

• no membrane channel is strictly necessary for sensing NO
• membrane pores / secretion channels / controlled membrane leakage for IL-10 emission: α-hemolysin nanopores?

3. a) Specific substances Lipids

   Lipid	Role
   POPC (phosphatidylcholine)	Main bilayer structure
   Cholesterol	Stability and reduced leakage
   DOPE	Improves membrane fluidity
   Cardiolipin	Stabilizes membrane proteins

Genes:

• norR (NO receptor)
• PnorV (NO-resp. promoter)
• human IL10
• sfGFP reporter
• hla (S. aureus α-hemolysin)
• groEL/groES/dnaK shsperones

3. b) Measuring system function

• sfGFP fluorescence measurement
• IL-10 ELISA assay
• membrane integrity assays (flow cytometry / microscopy)
• NO dose-response curve (RT-qPCR and Western blot assays for srecific genes/proteins)
• Functional inflammation assay (cytokine profiles)

HW question from Peter Nguyen

1. One sentence pitch

A “living fragrance textile” that uses freeze-dried cell-free systems embedded in fashion fabrics to sense body chemistry and dynamically produce personalized perfume molecules throughout the day.

How the idea works

The concept is a smart fashion fabric containing microencapsulated freeze-dried cell-free transcription/translation (Tx/Tl) systems integrated into clothing, scarves, jewelry textiles, or wearable accessories. These systems remain dormant while dry but become activated when exposed to moisture from sweat, humidity, or skin contact. The activated cell-free biosensors detect biochemical signals such as skin pH, stress metabolites, temperature changes, or sweat composition and respond by enzymatically generating fragrance compounds directly within the textile.

For example: Increased sweat/stress biomarkers ↓ Rehydration of freeze-dried Tx/Tl system ↓ Expression of fragrance-producing enzymes ↓ Localized release of perfume molecules

Customization would likely become one of the most attractive features of the system. The textile could create various scents during stress, exercise, or related with outer temperature.

Different scent molecules are produced by different enzymatic pathways:

This is conceptually realistic because:

• cell-free systems are modular
• DNA templates are easily replaceable
• fragrance synthesis pathways already exist in plants and microbes
• freeze-dried reactions can be pre-programmed

The major challenge is not sensing itself, but:

• stability
• precise dosage control
• long-term scent consistency
• scalable manufacturing

Societal challenge or market need

1. Personalized fragrance experiences Traditional perfumes are static and identical for all users, despite body chemistry strongly affecting scent perception. A responsive biosynthetic textile could create individualized fragrances that adapt in real time to:

• body chemistry
• stress
• environment
• activity level

This enables hyper-personalized fashion experiences.

2. Sustainable perfumery Conventional perfume manufacturing often involves:

• petrochemical synthesis
• excessive packaging
• solvent-heavy production
• environmentally intensive ingredient sourcing

Cell-free fragrance synthesis could reduce overproduction, waste, and transportation of volatile compounds. The textile would produce scent only when needed.

3. Long-lasting wearable fragrance Perfumes typically evaporate rapidly and require reapplication. A cell-free fragrance textile could continuously regenerate scent molecules over time, extending fragrance longevity without carrying additional products.

4. Emotional wellness and sensory fashion Fashion is increasingly integrating wellness and multisensory design. Adaptive scent-producing textiles could:

• reduce stress
• improve mood
• enhance confidence
• create immersive sensory experiences

Thus, the idea escapes narrow fashion context with luxury wearables, and performance costumes, being theoretically expandable onto therapeutic fashion and wellness accessories.

Addressing limitations of cell-free systems

1. Moisture-triggered activation Sweat and humidity naturally serve as activation mechanisms:

Dry textile → dormant biosystem Skin moisture/sweat → activation

2. Protective encapsulation for stability Heat, oxidation, and UV exposure are primary sources of Tx/Tl components destabilization of wearable freeze-dried systems. They could be encapsulated in:

• silk fibroin microcapsules
• hydrogel nanofibers
• trehalose sugar matrices

These materials are also compatible with textiles and fashion materials.

3. Replaceable fragrance patches Rather than washing the biological system repeatedly, garments could contain:

• detachable fragrance inserts
• replaceable biosensing textile modules
• refillable collar or cuff patches

This makes the fashion piece reusable while replacing only the active biological component.

4. Controlled one-time or slow-release activation The system could be designed for single-event luxury scent release or gradual activation over hours. Hydrogels and layered polymer barriers could regulate:

• water diffusion
• enzyme activation
• fragrance release rate

5. Stabilizing the energy supply The freeze-dried system would include:

• ATP regeneration enzymes
• glucose or maltodextrin energy reservoirs
• slow-release substrates.

This could extend fragrance production during wear.

6. One-time use as a luxury feature In haute couture or perfumery, ephemerality can become part of the artistic concept and experience. For example:

• a dress that releases a unique fragrance only during a runway performance
• a perfume scarf activated once during an event
• personalized scent “moments” linked to environmental conditions

The transient nature of cell-free reactions could therefore enhance exclusivity rather than limit functionality.

HW question from Ally Huang

Background

In space, astronauts face increased exposure to cosmic radiation and microgravity, which can damage DNA and disrupt cellular function. This raises risks such as cancer, immune dysfunction, and accelerated aging. Monitoring DNA damage in real time is challenging due to limited lab infrastructure aboard spacecraft. Freeze-dried cell-free systems like BioBits® provide a safe, stable, and portable platform for biological sensing without using living cells. These systems can be deployed on-demand and rehydrated when needed. Developing reliable DNA damage sensors is important for astronaut safety and for understanding how biological systems respond to extreme environments beyond Earth.

Molecular or genetic target

Escherichia coli recA promoter–GFP reporter construct (SOS DNA damage response pathway)

Relation to the space biology

The recA promoter is part of the bacterial SOS response, which activates when DNA damage occurs due to radiation stress. In the BioBits® cell-free system, a plasmid containing the recA promoter linked to GFP is added after rehydration. Importantly, the system does not interact with human DNA; instead, it detects environmental stress signals by responding only to the engineered DNA template. When DNA damage conditions are simulated, activation of the promoter leads to GFP production. This provides a controlled, indirect biosensing method for estimating radiation-related stress in space-relevant conditions.

Hypothesis

The hypothesis is that a freeze-dried BioBits® cell-free system containing a recA promoter–GFP reporter will show significantly higher fluorescence when exposed to DNA-damaging conditions compared to non-damaged controls. This is based on the principle that the recA promoter is activated during the SOS response to DNA damage, leading to increased downstream gene expression. In our system, GFP production acts as a direct, measurable output of promoter activation. When exposed to simulated space radiation stress, such as UV light, experimental samples should exhibit increased fluorescence intensity relative to controls. This would demonstrate that cell-free biosensors can reliably detect genotoxic stress. The results would support the use of BioBits®-based systems for monitoring astronaut exposure to radiation and for developing autonomous, low-resource diagnostic tools for long-duration space exploration missions.

Experiment design

Freeze-dried BioBits® reactions will be prepared containing a recA promoter–GFP plasmid. Samples will be rehydrated and divided into experimental and control groups. Experimental groups will be exposed to UV light as a radiation proxy, while controls remain unexposed. The miniPCR® thermal cycler will be used to amplify DNA constructs if required before reaction setup. After incubation, GFP fluorescence will be measured using the P51 Molecular Fluorescence Viewer. Negative controls will include reactions without plasmid DNA. Fluorescence intensity will be compared across conditions to quantify activation of the DNA damage response biosensor.