Week 9 HW: Cell-Free Systems

General homework questions

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.

• Direct control of conditions: You can independently adjust pH, temperature, ion concentration, redox state, and cofactors without affecting cell viability.
• Faster optimization cycles: No need for cloning, transformation, or cell growth—results can be obtained in hours.
• No cellular constraints: Toxic proteins, unstable proteins, or proteins that burden cells can still be produced.
• Simplified system: Fewer regulatory pathways means fewer unknown biological interactions.

Cases where cell-free is more beneficial:

• Expression of toxic proteins
• Rapid protein prototyping
• Production of proteins requiring non-standard conditions or non-natural amino acids.

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

Main components of a cell-free expression system and their roles:

• Cell extract (lysate): Provides ribosomes, tRNAs, aminoacyl-tRNA synthetases, and translation machinery.
• Cell-free protein synthesis template DNA or mRNA: Encodes the protein of interest.
• Energy system: Supplies ATP/GTP for transcription and translation.
• Amino acids: Building blocks for protein synthesis.
• Salts and ions (Mg²⁺, K⁺): Stabilize ribosomes and enzyme activity.
• Buffer system: Maintains optimal pH.

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.

Protein synthesis is extremely energy-intensive: ATP and GTP are required for transcription, translation, tRNA charging, and elongation steps. Without regeneration, the system quickly stalls.

In Cell-free protein synthesis systems, ATP depletion is one of the main limiting factors.

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

Prokaryotic system

• Fast, cheap, high yield
• Best for simple proteins without complex modifications

Example protein: Green fluorescent protein (GFP) Reason: folds easily, no glycosylation required, expresses efficiently

Eukaryotic system

• Supports post-translational modifications
• Better for complex human proteins

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.

Designing a cell-free system for membrane protein expression:

• Include detergents or nanodiscs/liposomes to mimic membranes
• Use oxidizing conditions if disulfide bonds are needed
• Add molecular chaperones to assist folding
• Optimize magnesium and potassium concentrations

Key challenges:

• Aggregation of hydrophobic regions
• Misfolding due to lack of membrane insertion
• Low solubility

Homework question from Kate Adamala

1. Function: Biosensing of glucose with fluorescent output

Synthetic cell detects glucose in the environment and produces a fluorescent signal (eGFP). Input: glucose Output: fluorescence (GFP signal) Partially, yes. A cell-free transcription/translation (Tx/Tl) system can produce GFP in response to glucose in solution. However, without encapsulation, the system lacks compartmentalization, stability, and control over the local environment. Yes, this function could be implemented in a genetically engineered bacterium such as Escherichia coli. The system should produce a fluorescence signal proportional to glucose concentration, with:

• low background signal
• high sensitivity
• fast response

2. Membrane

The synthetic cell will be a liposome composed of:

• phosphatidylcholine (POPC)
• cholesterol (for stability)

Tx/Tl system - cell-free extract Genetic circuit - glucose-responsive promoter, reporter gene: eGFP and egulatory system: glucose-sensitive transcription factor

A bacterial system (E. coli) is sufficient because:

• no complex post-translational modifications are required
• efficient and widely used

3. List of components

Lipids:

• POPC
• cholesterol

Genes:

• eGFP (reporter)
• glf (glucose transporter)

How to measure function:

• fluorescence spectroscopy
• plate reader measurements
• fluorescence microscopy

Homework question from Peter Nguyen

Integrate freeze-dried Cell-free protein synthesis systems into building materials to create pollution-responsive facades that actively detect and degrade urban air contaminants.

The proposed system embeds freeze-dried cell-free expression components directly into construction materials such as concrete panels or surface coatings. These systems contain DNA templates encoding enzymes capable of degrading airborne pollutants (e.g., NOx or volatile organic compounds).

Societal challenge. Urban air pollution remains a major global challenge, particularly in densely populated cities where traditional mitigation strategies are limited. Current architectural materials are largely passive and do not contribute to environmental remediation.

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

Activation (water dependence). The system is designed to be moisture-activated, using naturally occurring environmental water sources such as rain or humidity, eliminating the need for external energy input.

Homework question from Ally Huang

Вackground

Long-duration space missions expose astronauts to microgravity and increased radiation, which can alter microbial behavior and increase the risk of pathogenicity and antibiotic resistance. Monitoring microbial gene expression in real time is critical for crew health and spacecraft safety. Traditional cell-based assays are difficult to maintain in space due to resource limitations. Freeze-dried Cell-free protein synthesis systems, such as BioBits®, offer a stable, low-resource alternative for detecting specific genetic signatures. This project proposes a portable, rapid-response system to monitor microbial stress responses during spaceflight.

Molecular Target

Stress-response and virulence-associated genes from spacecraft-associated bacteria such as Escherichia coli.

Relevance of Target

Stress-response genes such as recA and rpoS are upregulated under DNA damage and environmental stress, conditions common in space. Monitoring their expression provides insight into how microbes adapt to microgravity and radiation. Increased expression of virulence-related genes (e.g., toxA) may indicate elevated pathogenic potential, posing risks to astronaut health. By targeting these genes, the system can function as an early-warning biosensor, enabling detection of harmful microbial shifts before they lead to infection or system contamination.

Hypothesis

Microgravity and space-related stressors induce measurable increases in microbial stress-response gene expression, which can be detected using a freeze-dried cell-free system. Specifically, BioBits® reactions programmed with DNA templates responsive to target gene sequences will produce a fluorescent signal when these genes are present or amplified. The goal is to develop a rapid, portable biosensing platform that integrates DNA amplification with cell-free protein expression and fluorescence detection. This system would enable astronauts to monitor microbial adaptation in real time without the need for complex laboratory infrastructure.

Experimental Plan

Bacterial samples will be processed to extract DNA. Target genes will be amplified using miniPCR®. Amplified DNA will be added to BioBits cell-free reactions containing reporter constructs that produce fluorescence upon detection. Fluorescence output will be measured using the P51 Molecular Fluorescence Viewer. Controls include:

• negative control (no DNA);
• positive control (known target DNA) Data will consist of fluorescence intensity over time, indicating gene presence and relative activity.