Week 9 HW: Cell Free Systems

Part A: General and Lecturer-Specific Questions

General

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.

In vitro transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. It is much easier to modify conditions and fine-tune levels of concentration. ¹

1. Toxic Protein Production Proteins that are toxic to living cells (e.g., membrane-disrupting proteins or toxins) can be safely produced In in vivo systems, these would kill the host cells before sufficient protein is made

2. Rapid Prototyping / Synthetic Biology Useful for quickly testing genetic constructs (e.g., promoters, circuits) without cloning and culturing Ideal for iterative design workflows

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

Cell-free expression works through the coupling of transcription (TX) and translation (TL) inside of a test tube.

1. Transcription from a DNA template to a mRNA -> RNA polymerase
2. Translation from RNA into protein(s) -> ribosome complex + tRNA

Main Components

Cell extract (lyse): It is composed of the molecular machinery and co-factors need for reactions

• ribosome ribonucleic complex
• RNA polymerase
• other desired proteins

tRNA: Transfers specific amino acids to the ribosome during protein synthesis by matching codons with anticodons.

polymerase: An enzyme (e.g., RNA polymerase) that synthesizes RNA from a DNA template during transcription.

nucleotides: Building blocks of nucleic acids that are used to construct RNA (and DNA).

folic acid: Acts as a cofactor in one-carbon metabolism, supporting nucleotide synthesis and overall metabolic activity.

coenzyme A: Functions as an acyl group carrier, playing a key role in energy metabolism and biochemical reactions.

3-PGA: Serves as an energy source in some cell-free systems by helping regenerate ATP.

RNA template: Provides the direct sequence information for protein synthesis during translation.

hepes buffer: Due to its high solubility, low membrane permeability, and negligible metal ion binding, it is considered a “Good’s” buffer ideal for optimizing biochemical reactions.

spermidine: Stabilizes nucleic acids and enhances transcription and translation efficiency.

sodium oxalate: Acts as a chelating agent that can influence ion balance in the reaction mixture.

NAD: A cofactor involved in redox reactions, supporting metabolic processes and energy balance.

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.

Cell-free systems lack metabolism, so they cannot naturally regenerate ATP. However, protein synthesis is highly energy-demanding.

One common strategy is to use 3-PGA as an energy source: 3-PGA is metabolized by enzymes present in the cell extract. This generates ATP from ADP through endogenous metabolic pathways. Instead of adding a fixed amount of ATP (which is quickly depleted), energy regeneration systems like 3-PGA ensure a continuous ATP supply, enabling longer and more efficient protein synthesis in cell-free systems. benefits

• Provides a slow, sustained release of energy
• Reduces accumulation of inhibitory byproducts compared to direct ATP addition
• Extends the reaction time and increases protein yield

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

Prokaryotic systems (e.g., Escherichia coli lysate) Advantages:

• Fast protein synthesis
• High yields
• Cost-effective and simple to use Limitations:
• Lack of post-translational modifications (PTMs) like glycosylation
• Limited ability to correctly fold complex eukaryotic proteins

Green Fluorescent Protein (GFP): It doesn’t require complex PTMs

Eukaryotic systems (e.g., wheat germ, insect, or mammalian lysates) Advantages:

• Capable of post-translational modifications (e.g., glycosylation, disulfide bonds)
• Better folding of complex proteins Limitations:
• More expensive
• Slower protein production
• Typically lower yields

Human Insulin: It requires correct disulfide bond formation

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.

Challenges Hydrophobic regions → cause aggregation or precipitation Lack of membrane → improper folding and loss of function Low solubility → reduced yield Complex structure → requires correct insertion and orientation

To optimize membrane protein expression in a cell-free system, I would design an experiment that recreates a membrane-like environment while systematically controlling reaction conditions. A key challenge is that membrane proteins contain hydrophobic regions, which can lead to aggregation and misfolding in the absence of a lipid bilayer. To address this, I would supplement the system (e.g., based on Escherichia coli lysate) with membrane mimetics such as detergents, liposomes, or nanodiscs to enable proper folding and insertion. Additionally, I would optimize parameters like ion concentrations, temperature, and DNA levels, and include chaperones to improve protein stability. By running parallel reactions with different conditions, I could identify the setup that maximizes soluble and functional protein yield.

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.

One possible reason is insufficient or degraded DNA template, which limits transcription. This can be addressed by checking DNA quality (e.g., avoiding degradation), increasing template concentration, or using a stronger promoter to enhance transcription efficiency.

A second reason could be inefficient energy supply, leading to early termination of protein synthesis. Since cell-free systems cannot regenerate ATP naturally, adding or optimizing an energy regeneration system (e.g., using 3-PGA or PEP) can help sustain longer and more productive reactions.

A third issue may be protein misfolding or aggregation, especially for complex or hydrophobic proteins. This can be improved by lowering the reaction temperature, adding chaperones, or including membrane mimetics (for membrane proteins) to promote proper folding and increase the amount of soluble, functional protein.

Homework question from Kate Adamala

Design an example of a useful synthetic minimal cell as follows:

1. Pick a function and describe it.

The synthetic cell produces natural pigments that could be used to dye cotton fibers sustainably directly on fibers.

2. hat would your synthetic cell do? What is the input and what is the output?

Function: Biosynthesis of plant-based pigments (e.g., anthocyanins) Input: Simple nutrients (glucose) + an inducer molecule (e.g., light or small molecule) Output: Visible color (e.g., red/purple pigment)

3. Could this function be realized by cell-free Tx/Tl alone, without encapsulation? Could this function be realized by genetically modified natural cell?

Cell-free Tx/Tl alone? Partially yes — pigment enzymes can be expressed in a cell-free system, but:

• Yield and stability are limited
• No compartmentalization → less control

Genetically modified natural cell? Yes, very feasible

• Plants or microbes already produce pigments naturally
• But less controllable and less “designable” than synthetic cells

4. Describe the desired outcome of your synthetic cell operation. A self-contained synthetic vesicle that:

• Produces visible pigment when triggered
• Could be applied to cotton fibers
• Enables localized, low-waste dyeing

5. Design all components that would need to be part of your synthetic cell. What would be the membrane made of? What would you encapsulate inside? Enzymes, small molecules. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian)

Membrane: Lipid bilayer (liposomes) Internal components:

• Tx/Tl system
• Genes for Pigment Production: PAL, CHS, F3H, DFR, ANR, and UFGT ² Small Molecules & Cofactors
• Glucose → energy source
• ATP regeneration system (e.g., 3-PGA)
• NADPH → required for biosynthesis
• Salts & nucleotides

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

Challenge: Liposomes are semi-permeable but limited

-> Solutions: Passive diffusion of small molecules (e.g., glucose) OR express membrane channels:

Example:

α-hemolysin (αHL) pore protein → allows small molecules to enter/exit

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

Genes: PAL, CHS, F3H, DFR, ANR, and UFGT

Lipids Phosphatidylcholine (PC) Phosphatidylglycerol (PG) Cholesterol

8. How will you measure the function of your system?

1. Color Output Visual observation (color change) Spectrophotometry (absorbance ~520–550 nm for anthocyanins)
2. Protein Expression SDS-PAGE or fluorescence tagging
3. Efficiency Pigment concentration over time
4. Application Test Dye uptake on cotton fibers Wash stability

Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

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

A swimwear garment embedded with freeze-dried cell-free biosensors that activate upon water exposure and produce a visible signal indicating unsafe water quality.

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

The bikini is made from textiles integrated with microencapsulated, freeze-dried BioBits® cell-free systems. These systems contain DNA constructs encoding fluorescent or color-producing reporter proteins under the control of contaminant-responsive genetic circuits. When the bikini is immersed in water, the embedded systems rehydrate and become biologically active. If contaminants such as bacteria, toxins, or chemical pollutants are present, the genetic circuit is activated and triggers the expression of the reporter protein. This results in a visible color change or fluorescence in specific areas of the fabric, indicating that the water quality may be unsafe. Different regions of the garment could be engineered to respond to different types of contaminants, enabling a multi-signal readout.

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

This concept addresses the growing need for personal, real-time environmental monitoring in recreational and natural water bodies such as lakes, rivers, and oceans. Water pollution can pose health risks that are not always visible, and current testing methods are often not accessible to individuals at the point of use. A wearable biosensor provides immediate feedback, empowering users to make informed decisions about water safety. It also aligns with increasing demand for smart, functional, and sustainable textiles that integrate biological sensing without electronics.

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

Activation with water: The system is intentionally designed to activate upon immersion, using water as the trigger to rehydrate the freeze-dried components. Stability: Freeze-drying preserves the cell-free reactions during storage, while encapsulation within protective polymer or hydrogel microdomains embedded in the fabric prevents premature activation and degradation. One-time use: The biosensor could be designed as a semi-consumable feature, where certain zones activate per exposure, or the garment could include replaceable sensing patches. Environmental robustness: Encapsulation also helps protect the system from mechanical stress, UV exposure, and repeated washing until activation is desired.

Homework question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space!

For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/ .

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)

In space missions, astronauts depend on closed-loop water recycling systems, making water quality monitoring critical for survival. Contaminants such as microbial byproducts, toxins, or chemical impurities can accumulate without easy detection. Traditional laboratory testing is impractical in space due to limited equipment, time, and resources. A portable, cell-free biosensor based on BioBits® offers a lightweight, stable, and on-demand solution for detecting water contamination. This approach is significant for ensuring astronaut health, enabling safe long-duration missions, and advancing compact diagnostic technologies that are also relevant for water quality monitoring on Earth in remote or resource-limited environments.

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)

A contaminant-responsive genetic circuit encoding a fluorescent reporter protein under the control of a promoter activated by bacterial toxins or stress-inducible regulatory elements.

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

The molecular target is directly linked to detecting harmful substances in recycled spacecraft water. In a cell-free BioBits® system, the genetic circuit is designed so that the presence of contaminants activates transcription and translation of a fluorescent reporter gene. This fluorescence serves as a measurable output indicating contamination levels. By coupling contaminant-sensitive regulatory elements to reporter expression, the system translates otherwise invisible molecular signals into a detectable readout. This allows rapid, in situ assessment of water safety without the need for complex instrumentation, addressing the critical need for reliable environmental monitoring in space habitats.

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

We hypothesize that a BioBits® cell-free system containing a contaminant-responsive genetic circuit will produce a measurable fluorescent signal in response to contaminated water samples, while remaining inactive in clean water. Specifically, regulatory elements sensitive to bacterial components or stress-related molecules will activate expression of a fluorescent reporter protein when contaminants are present. The intensity of fluorescence will correlate with contaminant concentration, enabling semi-quantitative assessment of water quality. The reasoning behind this hypothesis is that cell-free transcription and translation machinery can be programmed with DNA constructs to function as biosensors, converting specific molecular inputs into optical outputs. Demonstrating this capability would show that freeze-dried cell-free systems can serve as reliable, portable diagnostic tools for monitoring water safety in space environments where conventional testing methods are not feasible.

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)

BioBits® freeze-dried cell-free reactions will be prepared with DNA constructs encoding a fluorescent reporter under a contaminant-responsive promoter. Test samples will include clean water (negative control), water spiked with simulated contaminants (e.g., bacterial lysate or chemical analogs), and reactions without DNA (background control). Water samples will be added to activate the reactions, followed by incubation. Fluorescence will be measured using the P51 Molecular Fluorescence Viewer. Fluorescence intensity will be compared across conditions to evaluate sensitivity and specificity of the biosensor in detecting contamination levels.