Week 9: Cell-Free Systems

Assignment Part A: General and Lecturer-Specific Questions

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.

You can control everything directly. You can adjust pH, temperature, ion concentrations, and cofactors and even add or remove components during the reaction. Also, it’s faster since you don’t need cell growth or maintenance.

In terms of control, you can precisely tune the system and avoid cellular regulation that might limit expression.

Cases where it’s more beneficial:

• Expression of toxic proteins, because they would kill the cells
• Rapid protein production for screening experiments, testing many variants quickly

2. Describe the main components of a cell-free expression system and explain the role of each component.

• Cell extract: Provides the machinery (ribosomes, tRNAs, enzymes) for transcription and translation
• DNA or mRNA template: Contains the gene for the protein to be expressed
• Amino acids: Building blocks for the protein
• Energy system (ATP, GTP): Powers transcription and translation
• Buffers and salts (Mg²⁺, K⁺): Maintain optimal conditions for the reaction

3. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

Because energy systems are consumed very quickly during transcription and translation. If it runs out, protein synthesis stops.

To maintain ATP levels, it’s possible to use phosphoenolpyruvate (PEP). It works by transferring a phosphate group to ADP, continuously regenerating ATP during the reaction.

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

• Prokaryotic systems are faster, cheaper, and give high yields, but they lack complex post-translational modifications.

• Eukaryotic systems are slower and more expensive, but they allow proper folding and modifications like glycosylation.

Examples:

Prokaryotic: GFP, because it’s simple and doesn’t need modifications Eukaryotic: an antibody, because it requires correct folding and post-translational modifications to function properly

5. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup.

• To mimic the membrane: Add liposomes or nanodiscs
• Keep the protein soluble: Use mild detergents
• Adjust conditions like Mg²⁺, temperature, and protein concentration
• Include chaperones to help folding of the protein

Challenges and how to adress:

• Misfolding: Membrane proteins don’t fold well without lipids → Solution: add liposomes or nanodiscs + chaperones

• Aggregation: Hydrophobic regions stick together → Solution: use mild detergents and lower expression rate (e.g., lower temperature)

• Low solubility/insertion efficiency → Solution: optimize lipid composition and Mg²⁺/salt conditions

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.

• Poor DNA/template quality or design → Troubleshooting: use a stronger promoter, optimize codons, check DNA purity
• Energy depletion → Troubleshooting: improve the energy regeneration system (e.g., add PEP or increase substrates)
• Protein misfolding or degradation → Troubleshooting: add chaperones, lower temperature, optimize reaction conditions

Assignment Part A: question from Kate Adamala

1. Pick a function and describe it.

• What would your synthetic cell do?

It would act as a diagnostic biosensor for the detection of DENV. Its primary function is to transduce the presence of the viral E protein into a detectable signal, utilizing a two-stage recognition system:

External Sensor: A specific antibody and aptamer recognizes the DENV E protein, this recognition event triggers the release of theophylline, which acts as a messenger molecule.

Internal Sensor (Synthetic cell): The theophylline binds to a theophylline riboswitch, inducing a conformational change that exposes the ribosome binding site (RBS), allowing the translation of the LacZ reporter gene, generating a detectable signal.

What is the input and what is the output? (synthetic cell)

Input: Theophylline released by the external sensor

Output: Expression of beta-galactosidase (LacZ)

• Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

Yes. This process can be carried out using only a cell-free Tx/Tl system. In this simplified setup, theophylline diffuses freely to activate LacZ expression without the need for encapsulation.

• Could this function be realized by genetically modified natural cell?

It’s possible, but a cell-free system is more economical and streamlined than traditional cell-based methods for this purpose.

• Describe the desired outcome of your synthetic cell operation.

The primary objective is to develop a sensitive, specific, user-friendly, and cost-effective biosensor for DENV detection. BUT the desired outcome of the synthetic cell operation is the expression of beta-galactosidase (LacZ) in the presence of theophylline

2. Design all components that would need to be part of your synthetic cell.

• What would be the membrane made of?

Phospholipid liposomes and polymersomes to ensure membrane stability, permeability, and biocompatibility

• What would you encapsulate inside? Enzymes, small molecules.

Genetic circuit, E. coli extract for Tx/Tl machinery and chromogenic substrates for β-galactosidase

• Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason?

E. coli

• How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

Utilizing an alpha-hemolysin channel to ensure the diffusion of theophylline and ONPG (the substrate for beta-galactosidase)

3. Experimental details

• List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)

Lipids: POPC, Cholesterol, DSPE-PEG2000 Enzymes: E.Coli bacterial cell-free Tx/Tl Genes: alpha-hemolysin, LacZ

• How will you measure the function of your system?

System functionality is quantified through beta-galactosidase-mediated hydrolysis of ONPG

Assignment Part A: question from Peter Nguyen

FirstIdea

1. Write a one-sentence summary pitch sentence describing your concept

Walls that use cell-free photosynthetic systems integrated into the material to capture CO₂ from the air and transform it locally into oxygen during daily sun exposure.

2. How will the idea work, in more detail? Write 3-4 sentences or more.

The building’s exterior walls incorporate modular layers containing cell-free systems based on artificial or reconstituted photosynthetic pathways. These modules are activated by sunlight and controlled ambient humidity, triggering biochemical reactions that capture CO₂ from the air and release oxygen as a byproduct

3. What societal challenge or market need will this address?

The urgent need to reduce CO₂ in dense urban environments, it proposes an architecture that not only minimizes its footprint but also actively acts as environmental infrastructure, which is especially relevant in cities with high pollution and constant solar exposure.

4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Activation is controlled by micro-encapsulation, which regulates water ingress, preventing unwanted continuous activation. For stability, stabilizers and housed in replaceable cartridges within the façade, allowing for periodic maintenance. By acknowledging the one-time or limited-cycle nature of these reactions, the idea is treat them as consumable layers—similar to filters—integrated into a building renewal cycle without compromising its main structure.

SecondIdea

1. Write a one-sentence summary pitch sentence describing your concept

An eco-friendly, ’living’ window coating utilizing freeze-dried cell-free systems to express UV-active chromoproteins that create avian-visible warning patterns while remaining perfectly transparent to the human eye.

2. How will the idea work, in more detail? Write 3-4 sentences or more.

A cell-free platform embedded into a transparent, porous biopolymer matrix applied to glass surfaces. When activated, the machinery expresses specialized UV-chromoproteins or enzymes that produce pigments with high absorbance/reflectance in the $300\text{–}400\text{ nm}$ range, which falls within the tetrachromatic visual spectrum of birds. These biological “inks” are arranged in specific geometric patterns that birds recognize as solid obstacles, triggering an avoidance response. Because humans lack UV photoreceptors and the proteins do not scatter visible light, the window appears clear to us while appearing “solid” or patterned to birds.

3. What societal challenge or market need will this address?

Every year, it is estimated that up to one billion birds die just in the United States alone due to collisions with glass windows, making it a leading cause of avian mortality and biodiversity loss. While UV-reflective stickers and “fritted” glass exist, they are aesthetically unpleasing, or lose effectiveness over time. There is a significant market need for sustainable, “invisible” retrofitting solutions for residential and commercial skyscrapers that can protect migratory species without altering architectural aesthetics

4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

• Stability: The cell-free machinery is freeze-dried within a protective matrix of trehalose or synthetic polymersomes, allowing it to remain dormant and stable during transport and installation.

• Activation: The system is designed for hydro-activation; ambient humidity or rain triggers the controlled release of water into the micro-compartments, initiating protein synthesis precisely when the risk of collision (often higher in overcast/rainy weather) is present.

• Longevity: To address the one-time use limit, the system would incorporate genetic circuits for protein stability and “slow-release” mechanisms where the chromoproteins are cross-linked to the matrix, ensuring the UV signal persists for several months before a simple, biodegradable “recharge” spray is required.

Assignment Part A: question from Ally Huang

1. Provide background information that describes the space biology question or challenge you propose to address. Explain why this topic is significant for humanity, relevant for space exploration, and scientifically interesting. (Maximum 100 words)

High‑energy heavy ions (HZE radiation) are a major hazard in deep‑space missions and are known to cause complex DNA and protein damage. Understanding how radiation accelerates molecular aging is essential for astronaut health, long‑duration missions, and space habitation. Cell‑free systems provide a lightweight, non‑living platform to directly study radiation‑induced molecular damage without confounding cellular repair mechanisms. This makes them ideal for spaceflight experiments, where resources are limited and biological containment is critical. Studying molecular aging in space has direct implications for human longevity, cancer risk, and the stability of biological systems beyond Earth.

2. Name the molecular or genetic target that you propose to study. Examples of molecular targets include individual genes and proteins, DNA and RNA sequences, or broader -omics approaches. (Maximum 30 words)

Green fluorescent protein (GFP)–encoding DNA and expressed GFP protein as molecular reporters of radiation‑induced damage and aging.

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

GFP is a well‑characterized protein whose fluorescence is sensitive to errors in DNA transcription, protein folding, and structural integrity. Damage from HZE radiation may reduce protein yield, alter folding efficiency, or degrade fluorescence intensity. Using GFP in a BioBits® cell‑free system allows direct measurement of radiation‑induced molecular aging without cellular repair or replication. Differences in expression level or fluorescence provide a clear molecular readout of how space radiation affects fundamental biological processes.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

Exposure to HZE radiation causes measurable molecular aging in cell‑free systems, leading to decreased protein expression efficiency and reduced fluorescence intensity. Cell‑free reactions lack DNA repair mechanisms and protein turnover, making them highly sensitive indicators of cumulative radiation damage. If HZE radiation accelerates molecular aging, irradiated GFP‑encoding DNA or protein synthesis machinery will produce less functional GFP compared to non‑irradiated controls. By comparing fluorescence output, this experiment will isolate the direct effects of space radiation on molecular stability and function, independent of living cells.

5. Outline your experimental plan—identify the sample(s) you will test in your experiment, including any necessary controls, the type of data or measurements that will be collected, etc. (Maximum 100 words)

Freeze‑dried BioBits® reactions containing GFP DNA will be exposed to HZE radiation and compared to Earth‑based and flight non‑irradiated controls. Reactions will be rehydrated simultaneously and incubated under identical conditions. GFP fluorescence intensity will be measured using the P51 Molecular Fluorescence Viewer. Controls include unexposed freeze‑dried reactions and reactions expressing pre‑folded GFP. Data will consist of fluorescence intensity and expression consistency across conditions.