Week 9 HW: Cell-free Systems

GENERAL HW 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.

CFPS transitioned from a “black box” scenario to an open controllable system, so flexibility and control are the main advantages here. This allows us to precisely manipulate the concentrations of amino acids, salts, and templates. It also allows for the addition of non-canonical amino acids or cytotoxic agents that would otherwise kill a living host. Cases where CFPS is more beneficial:

• Production of cytotoxic proteins, because proteins that disrupt membrane integrity or interfere with vital cellular processes (like certain toxins or antimicrobial peptides) cannot be produced in vivo.
• Rapid prototyping: CFPS bypasses the time-consuming steps of cloning, transformation and cell cultivation, reducing the cycle from days to hours.

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

A standard CFPS system consists of:

• Whole cell extract (lysate): provides the essential molecular machinery, like ribosomes, enzymes, initiation/elongation factors, etc.
• Energy solution: containing an energy source (like glucose or phosphoenolpyruvate) and a buffer system to maintain an optimal pH and ionic strength.
• Reaction mix: includes the DNA template (plasmid or PCR product), RNA polymerase, nucleotides and amino acids.

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.

Energy provision is critical because protein synthesis is metabolically expensive: for every peptide bond formed, multiple high-energy phosphate bonds are hydrolyzed. Without regeneration, the accumulation of inorganic phosphate inhibits the reaction, and the system reaches thermodynamic equilibrium quickly, ceasing production. A method to ensure continuous ATP supply could be a semi-continuous or continuous-exchange bioreactor, which uses a dialysis membrane to facilitate the constant diffusion of fresh substrates like ATP and NTPs into the reaction chamber, while simultaneously removing inhibitory metabolic byproducts, increasing the system’s productivity.

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

Prokaryotic systems (e.g. E. coli) are simpler and faster, being highly efficient for expressing smaller, robust proteins like GFP because they lack complex compartmentalization, allowing for rapid coupled transcription and translation. However, they do not have post-translational modifications or are very limited, so it is not possible to produce human or therapeutic proteins that require them. Instead, eukaryotic systems are preferred. Although they generally offer lower yields and involve more complex preparation, they provide the necessary machinery for folding and glycosylation. In a prokaryotic system, I would choose to produce a GFP because it is a robust, non-glycosylated protein commonly used as a reporter. The high yield of E. coli lysates makes it ideal for quick quantification. My eukaryotic choice would be Human Erythropoietin because it requires specific glycosylation patterns to be biologically active and stable in the bloodstream that only this eukaryotic cells can provide.

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.

Expressing membrane proteins in these systems could be challenging due to their hydrophobic nature, which leads to aggregation and precipitation in aqueous lysates. To overcome this, may be possible to add synthetic lipids or detergents to the reaction, which will provide a hydrophobic scaffold for the protein to insert into during translation. We could also use lysates enriched with specific chaperones (like DnaJ/DnaK) to assist in proper insertion.

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.

a. Template degradation: checking the purity of the DNA and ensuring the environment is RNase-free by adding RNase inhibitors to the mix and avoiding contamination. b. Magnesium imbalance: ribosome stability and polymerase activity are extremely sensitive to magnesium concentration, so it is possible to perform a Mg+2 titration to ensure there is no difference. c. Codon bias: if expressing a human gene in E. coli lysate, the “rare codons” could ruin translation. A solution would be to supplement the reaction with tRNAs for rare codons or use a codon-optimized sequence.

HW QUESTIONS FROM KATE ADAMALA

I would design a synthetic minimal cell (SMC) for pathogen detection and response to function as a “sentinel” to expand the therapeutic capacity against hospital-acquired infections, specifically targeting Pseudomonas aeruginosa. The input would be a Quorum Sensing molecule produced by P. aeruginosa called 3-oxo-C12-HSL, and the therapeutic output a Bacteriophage T4 Lysozyme (a potent antibacterial enzyme). A fundamental aspect of this design is the encapsulation of the cell-free machinery within a phospholipid bilayer. This function cannot be realized by cell-free transcription and translation alone. Without the liposome membrane, both the genetic circuit amd the synthesized enzymes would be immediately diluted, preventing the high macromolecular crowding required for efficient reaction kinetics. The membrane acts as a diffusion barrier and a protective shield, preserving the interal system from exogenous proteases and nucleases commonly found in clinical environments, which would otherwise degrade the system before it reaches the pathogen. To implement this the SMC encapsulates an E. coli-derived PURE (Protein synthesis using recombinant elements) system and a specialized genetic circuit, including the lasR gene for constitutive sensor expression and the genes for α-hemolysin (aHL) and T4 lysozyme, both controlled by the PlasI promoter. This setup allows a great communication strategy: while the membrane is naturally permeable to the small 3-oxo-C12-HSL input molecule, the large lysozyme output can only exit the SMC through the aHL pores, which are synthesized only after the pathogen signal is detected. This ensures a localized and triggered response, effectively creating a “smart pill” that remains dormant until it senses a high concentration of the target pathogen. Finally, the success of this synthetic system will be measured by its ability to inhibit bacterial growth. In an experimental setting, I would monitor the optical density of P. aeruginosa cultures in the presence of the SMCs. A significant reduction in growth, confirmed by fluorescence assays, would demonstrate this system successfully detected the quorum-sensing signal and released a sufficient concentration of lysozyme to neutralize the infection.

Lipids and genes:

• Lipids: POPC, cholesterol
• Enzymes: bacterial cell-free Tx/TI (PURE System)
• Genes: lasR (constitutive expression of the transcription activator)
  • Α-hemolysin (aHL) under the PlasI promoter
  • T4 Lysozyme under the PlasI promoter
• Target cells: P. aeruginosa (WT)

HW QUESTIONS FROM PETER NGUYEN

I would choose to design a smart athletic textile integrated with these cell-free systems that act as a real-time metabolic sensor, providing a visible colorimetric readout of lactic acid levels through sweat activation. The integration of this sensor addresses a critical need in sports physiology, because lactate serves as a vital biomarker to identify the lactate threshold, the point beyond which metabolic acidosis triggers muscle fatigue and performance decline. The fabric is manufactured by embedding a cell-free “master mix” directly into the fibers of a synthetic textile. To make it, I would use a genetic circuit where a specific transcription factor (like LldR) senses lactate. In the presence of high lactic acid levels in the user’s sweat, the circuit is activated to express a high-intensity chromoprotein. To ensure the reaction stayls localized, the cell-free components are encapsulated in micro-hydrogels added into the fabric. The user’s sweat acts as the rehydration agent, triggering the on-demand protein synthesis, providing a localized visual map of muscle fatigue directly on the system. This garment addresses the growing need for non-invasive, real-time physiological monitoring in both sports and physical therapy. Currently, measuring metabolic markers like lactate requires blood draws or expensive electronic sensors. This “living” textile provides a zero-power, lightweight and intuitive way for athletes to optimize their training intensity and for patients in rehabilitation to monitor its health through simple visual feedback. So, the activation will be through the user’s sweat as the natural trigger. The sensitivity of the circuit is tuned so that a baseline amount of moisture is required, preventing accidental activation by ambient humidity. Following Dr. Nguyen’s protocols, stability will be addressed using the cell-free mix supplemented with lyoprotectans like trehalose and sucrose, which form a glassy state around the proteins and DNA, allowing the garment to be stored at room temperature in a sealed package without losing activity. To address the “one-and-done” nature of cell-free systems, the grment is designed with replaceable bio-patches in high-sweat areas like the lower back or underarms. This hybrid approach combines a durable, washable textile with low-cost, disposable biological insert.

HW QUESTIONS 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)

Cosmic radiation is a primary barrier to long-term human spaceflight causing oxidative stress and DNA damage. Even though our own DNA machinery repairs the damage, the amount of radiation exposure accumulates during their time in space making it really challenging to alleviate. Monitoring this risks in real-time is vital for astronaut’s health. Currently, samples must often be returned to Earth for complex analysis. That’s why BioBits offers a portable, cell-free alternative to detect these threats in situ without the metabolic burden of maintaining live cell cultures. Developing a rapid, on-station diagnostic for radiation-induced molecular damage is significant for deep-space missions where immediate medical decision-making is essential for long-term health monitoring.

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)

Reactive oxygen species (ROS)-responsive promoter regulating expression of a GFP reporter protein.

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

ROS are key indicators of oxidative stress, which increases under space conditions. By designing a ROS-responsive genetic circuit in a cell-free system we can directly link oxidative stress levels to measurable GFP fluorescence. This allows real-time detection without requiring living cells. Monitoring ROS levels helps evaluate astronaut health risks. The simplicity and portability of the system makes it ideal for space missions, where traditional laboratory assays are not feasible.

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

The hypothesis is that a freeze-dried cell-free expression system incorporating a ROS-responsive promoter can reliably detect oxidative stress by producing a quanfifiable fluorescent signal. Specifically, higher ROS levels will activate the promoter leading to increased GFP expression. This approach is based on the principle that oxidative stress activates specific regulatory elements in biological systems. By coupling these elements to a reporter gene in a cell-free format we eliminate the need for living cells while preserving biological responsiveness. The goal of this project is to develop a simple, robust biosensor that can function in space conditions and provide rapid and interpretable results, enabling astronauts to monitor their oxidative stress levels in situ and serve as a model for detecting other environmental or physiological changes during space missions.

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 reactions containing the ROS-responsive GFP construct will be rehydrated with samples exposed to varying ROS levels (eg. Hydrogen peroxide). A negative control with no ROS and a positive control with a known ROS concentration will be included. Reactions will be incubated using the miniPCR device, and fluorescence will be measured with the P51 viewer. Data will be collected as fluorescence intensity over time. Inscreased fluorescence compared to controls will indicate a ppsitive detection. This setup allows quantitative comparison of oxidative stress levels under simulated space conditions.