Week 9 HW: Cell-Free Systems

Part A: General and Lecturer-Specific Questions

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.

Cell-free protein synthesis provides major advantages over traditional in vivo protein expression because the reaction occurs outside living cells, giving researchers direct control over reaction conditions and components. Variables such as DNA concentration, salts, energy substrates, cofactors, temperature, and additives can be adjusted independently without needing to maintain cell viability, allowing rapid optimization and faster experimental iteration. Another key advantage is flexibility: proteins can be expressed immediately after adding DNA templates, without time-consuming cloning, transformation, or cell culturing steps. Cell-free systems also allow incorporation of non-natural amino acids, toxic proteins, or synthetic circuits that would otherwise harm or interfere with living cells. Two important cases where cell-free expression is more beneficial than cell-based production are:

  1. Expression of toxic proteins — proteins that damage membranes, inhibit metabolism, or kill cells can often still be produced efficiently in cell-free systems because there is no living host to maintain.
  2. Rapid prototyping of genetic constructs or biosensors — cell-free systems enable fast testing of promoters, enzymes, fluorescent proteins, or metabolic pathways within hours rather than days, making them highly useful for synthetic biology and high-throughput design workflows.

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

A Cell-free protein synthesis system contains several core components that work together to synthesize proteins outside living cells:

  • Cell lysate (extract): The lysate is the main biological machinery of the system and is typically prepared from organisms such as E. coli. It contains ribosomes, tRNAs, aminoacyl-tRNA synthetases, translation factors, metabolic enzymes, and often RNA polymerase, all of which are required for transcription and translation.
  • DNA template: The DNA plasmid or linear DNA contains the gene encoding the target protein under a promoter such as T7. This serves as the instruction set for producing mRNA and ultimately the protein of interest.
  • Amino acids: Free amino acids are supplied as the building blocks used by ribosomes during protein synthesis.
  • Energy source / energy regeneration system: Molecules such as phosphoenolpyruvate (PEP), 3-PGA, or ATP regeneration substrates provide the energy needed for transcription, translation, and other enzymatic reactions over the course of the reaction.
  • Nucleotides (NTPs): ATP, GTP, CTP, and UTP are needed for RNA synthesis during transcription and also contribute energy for translation processes.
  • Salts and buffering agents: Components such as magnesium glutamate, potassium glutamate, and HEPES buffer maintain optimal ionic strength and pH. Magnesium is especially critical for ribosome stability and enzyme activity.
  • Cofactors and supplements: Additional molecules such as NAD, CoA, folinic acid, spermidine, or tRNAs help support enzyme function, metabolic balance, and efficient translation.
  • RNA polymerase (if not already in lysate): In T7-based systems, T7 RNA polymerase transcribes the DNA template into mRNA. Some lysates already contain this enzyme, such as BL21(DE3)-derived extracts.

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 cell-free protein synthesis consumes large amounts of ATP and GTP during transcription, translation, aminoacyl-tRNA charging, and protein folding, but unlike living cells the reaction cannot continuously regenerate energy through normal metabolism. Once ATP is depleted, protein synthesis slows or stops, so fluorescence output over a long incubation depends strongly on maintaining energy supply. One method is to include an energy regeneration system, such as 3-phosphoglycerate (3-PGA) or phosphoenolpyruvate (PEP), together with the enzymes in the lysate that convert these substrates into ATP. This continuously replenishes ATP during the reaction, allowing longer protein production and higher final fluorescence over the 36-hour incubation.

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

Prokaryotic cell-free systems (commonly based on E. coli lysates) are fast, inexpensive, and produce very high protein yields. They are ideal for expressing simple bacterial proteins or rapidly prototyping genetic circuits because they have efficient transcription and translation machinery and are easy to optimize. However, they generally lack advanced post-translational modifications such as glycosylation and may struggle with complex eukaryotic proteins. For a prokaryotic system, I would choose to produce sfGFP because it folds efficiently, matures rapidly, and does not require complex modifications. Its robustness makes it highly suitable for high-yield expression in E. coli-based cell-free reactions.

Eukaryotic cell-free systems (such as wheat germ, insect, or mammalian extracts) better support complex protein folding, disulfide bond formation, and post-translational modifications that are important for many human proteins. Although they are usually more expensive and produce lower yields, they are advantageous for expressing proteins that require native-like processing and functionality. For a eukaryotic system, I would choose a human monoclonal antibody fragment or another glycosylated therapeutic protein because proper folding and post-translational modifications are critical for biological activity. A eukaryotic cell-free system would better reproduce the cellular environment needed for correct structure and function.

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.

Membrane proteins are difficult to express because their hydrophobic regions often aggregate or misfold without a membrane environment. To optimize expression in a Cell-free protein synthesis system, I would test reactions containing liposomes, nanodiscs, or mild detergents to support proper insertion and folding. I would vary conditions such as Mg²⁺ concentration, temperature, DNA concentration, and chaperone addition while measuring both fluorescence and protein functionality. Lower temperatures and membrane mimics would likely improve soluble, functional protein yield by reducing aggregation and promoting correct folding.

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.

Homework question from Kate Adamala

Pick a function and describe it.

antibiotic-sensing reporter vesicle

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

The synthetic cell detects the antibiotic tetracycline in the environment. Input: tetracycline outside the vesicle. Output: green fluorescence from sfGFP expression inside the vesicle.

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

Yes, a bulk cell-free reaction could detect tetracycline, but encapsulation makes it more “cell-like” and allows compartmentalized sensing, which is useful for artificial-cell experiments.

Could this function be realized by genetically modified natural cell?

Yes. A bacterium could be engineered with a tetracycline-responsive promoter controlling GFP expression. However, living cells introduce growth, metabolism, toxicity, and regulatory complexity.

Describe the desired outcome of your synthetic cell operation.

When tetracycline is present, the synthetic cell produces GFP and becomes fluorescent. Without tetracycline, fluorescence remains low.

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

What would be the membrane made of?

The membrane would be a lipid vesicle, for example made from POPC and cholesterol. This creates a stable artificial membrane around the cell-free reaction.

What would you encapsulate inside? Enzymes, small molecules.
  • cell-free Tx/Tl mastermix
  • DNA encoding the reporter circuit
  • amino acids
  • NTPs
  • salts and buffer
  • ATP regeneration system
  • TetR regulatory protein or DNA encoding TetR
  • sfGFP reporter gene
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)

An E. coli cell-free system is sufficient because the circuit can use a bacterial tetracycline-responsive promoter. A mammalian system is not needed unless using mammalian promoters such as Tet-ON.

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

Tetracycline is small and can diffuse across membranes to some extent, but permeability may be limited. To improve input access, I would include a membrane pore such as α-hemolysin so small molecules can enter the vesicle.

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: main phospholipid for vesicle membrane
  • Cholesterol: improves membrane stability
  • Optional: PEG-lipid such as DSPE-PEG to reduce vesicle aggregation

Genes:

  • tetR: encodes the TetR repressor
  • P_tet-sfGFP: tetracycline-inducible promoter controlling sfGFP
  • hla from Staphylococcus aureus: encodes α-hemolysin pore, if membrane permeability needs improvement
How will you measure the function of your system?

I would measure GFP fluorescence over time using a plate reader or fluorescence microscope. The key comparison would be vesicles with tetracycline versus vesicles without tetracycline; successful function means tetracycline-treated vesicles show clearly higher fluorescence after incubation.

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:

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

A smart protective textile could use freeze-dried cell-free biosensors printed into fabric to detect harmful chemicals or pollutants and produce a visible color or fluorescence warning.

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

The textile would contain small printed patches of freeze-dried cell-free expression mix, DNA sensor circuits, and color-producing reporter proteins. When the fabric becomes wet from sweat, rain, or a small added water droplet, the cell-free system rehydrates and becomes active. If a target molecule, such as a toxic industrial chemical, pesticide, or air pollutant, is present, it triggers expression of a reporter such as GFP, mScarlet, or an enzyme that produces a visible pigment. The result would be a wearable, low-cost warning system for workers, soldiers, farmers, or people in polluted environments.

What societal challenge or market need will this address?

Many people are exposed to unsafe chemicals without immediate detection tools, especially in agriculture, factories, disaster zones, and low-resource settings. A sensor textile could provide fast, portable, and easy-to-read exposure information without needing electronics or laboratory equipment.

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

Because cell-free reactions need water, the sensor patches would be sealed in dry protective microcapsules or printed hydrogel spots that activate only when intentionally wetted or exposed to moisture. Stability could be improved by freeze-drying with protectants such as trehalose or sucrose. Since many cell-free sensors are single-use, the textile could include replaceable sensor patches, similar to disposable test strips integrated into reusable clothing.

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/ .

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)

Long-duration spaceflight exposes astronauts to radiation, microgravity, and closed habitats, all of which can increase cellular stress and DNA damage. Monitoring biological stress quickly and with limited equipment is important because astronauts cannot rely on full Earth-based laboratories during missions. This topic is significant for humanity because future Moon and Mars missions will require autonomous health monitoring. It is scientifically interesting because space conditions may change how DNA damage and repair pathways behave compared with Earth.

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)

p53 pathway stress response, represented by a synthetic DNA construct expressing fluorescent protein in response to DNA damage-related signaling.

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

The p53 pathway is strongly linked to DNA damage, radiation response, and cellular stress. In space, increased radiation exposure may cause DNA lesions that activate stress-response pathways. A simplified BioBits cell-free reporter cannot fully recreate a human cell, but it can model whether a designed genetic sensor circuit can convert a DNA damage-related input into a visible fluorescent output.

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

One hypothesizes that a freeze-dried BioBits cell-free reaction can be used as a simple space-compatible biosensor for DNA damage-related stress signals. If a DNA damage-mimicking input or regulatory DNA sequence is added to the reaction, the system should produce a stronger fluorescent signal than the negative control. The reasoning is that cell-free systems are lightweight, shelf-stable, and do not require living cells, making them useful for space biology experiments where resources, storage, and safety are constrained. The research goal is to test whether a cell-free fluorescent reporter could serve as a prototype for astronaut health or environmental radiation monitoring.

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)

I would prepare BioBits cell-free reactions containing a fluorescent reporter construct and test three conditions: no input negative control, positive control with constitutive fluorescent protein expression, and experimental reaction with the DNA damage-responsive sensor design. Reactions would be rehydrated with water and incubated. Fluorescence would be measured using the P51 Molecular Fluorescence Viewer over time, for example at 0, 6, 12, and 24 hours. The main data would be fluorescence intensity and time to visible signal. A successful sensor would show higher fluorescence in the experimental condition than the negative control.

Part B: Individual Final Project

Put your chosen final project slide in the appropriate slide deck following the instructions on slide 1

Done ;)

Submit this Final Project selection form if you have not already.

Done ;)

Begin planning how you will write your final project documentation based on these guidelines

Done ;)

Prepare your first DNA order and put it in the “Twist (MIT)” or “Twist (Nodes)” tab of the 2026 HTGAA Ordering: DNA, Reagents, Consumables spreadsheet, as appropriate.

Done ;)