Week 9 HW: Cell-Free Systems

Homework Part A: General and Lecturer-Specific Questions

General homework 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.

Synthesizing proteins outside living cells allows for more control over the environmental variables (pH, temperature, particular concentrations of ions and molecules, etc.), as the cell is not interacting with complex surroundings related to metabolic processes, resources or cofactors. This control is beneficial for experiments such as those that involve artificial aminoacids, since they can’t be assembled inside natural cells; or those that could involve toxic proteins that can’t be produced inside living natural cells.

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

  • DNA/RNA template: encodes the target protein; tells the system what to make
  • RNA polymerase: transcribes DNA into mRNA
  • Ribosomes: read the mRNA and build the protein
  • tRNAs: carry amino acids to the ribosome, matching each codon
  • Amino acids: the building blocks of the protein
  • Nucleotides (ATP, GTP, etc.): building blocks for RNA and energy for the reactions
  • Energy regeneration system: continuously regenerates ATP so the reaction doesn’t stop early
  • Cofactors/coenzymes (Mg²⁺, K⁺, etc.): stabilize ribosomes and support enzymatic activity
  • Buffer: maintains stable pH
  • Cell extract (lysate): the practical source of most of the already mentioned components; provides ribosomes, polymerases, and translation factors all at once

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.

Transcription and translation are processes that consume large amounts of ATP and GTP. Since there’s no living cell to continuously produce new energy, the reaction will stop as soon as the initial ATP runs out. Also, the phosphate byproducts that accumulate can actually inhibit the reaction.

A solution for this is to include 3-PGA in the reaction. The enzymes already present in the cell extract will convert 3-PGA into ATP over time, maintaining energy levels throughout the experiment so the reaction doesn’t stop.

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

Prokaryotic cell-free expression systems are cheaper, faster, and give higher yields; they are especially useful for simple proteins that don’t need post-translational modifications. For example, GFP (green fluorescent protein) is a protein that does not require post-translational modifications and is perfect for E.coli based cell-free expression systems.

Eukaryotic systems, on the other hand, are more expensive and slow, but support proper folding and post-translational modifications needed by more complex proteins. For example, a human hormone requires specific disulfide bonds and glycosylation to be functional, so an eukaryotic cell-free system is necessary.

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.

Membrane proteins are hydrophobic, so without a membrane-like environment they tend to fold incorrectly or clump together as they’re synthesized.

To avoid this, membrane mimics (liposomes, nanodiscs, detergents, etc.) could be directly added to the reaction so the protein has somewhere to insert as it’s being made. Then, I could run optimization experiments of varying type and concentration of the chosen membrane mimic and temperature, checking protein yield (via gel or western blot) and functionality (via an activity assay) to confirm the protein is folding correctly.

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.

  • The mRNA or the protein is being broken down by contaminating enzymes in the extract. To fix this, you could add RNase inhibitors to protect the mRNA, or use a protease-deficient extract.

  • The reaction is running out of energy too quickly. To fix this, you could optimize or add more energy regeneration system (for example, increase 3-PGA concentration or use a creatine phosphate system).

  • The DNA concentration is too low, the quality is poor, or the promoter isn’t efficient enough. To fix this, you could increase the DNA input, check the purity, and/or use a stronger or more appropriate promoter.

Homework question from Kate Adamala

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

1. Pick a function and describe it.

An example could be a synthetic minimal cell that detects an antibiotic and reports its presence, which could be useful to evaluate clinical or environmental samples. More specifically, the cell detects tetracycline, a broad-spectrum antibiotic, and produces a visible signal with GFP (green fluorescent protein) fluorescence.

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

Input: tetracycline Output: GFP fluorescence

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

Technically yes, because the sensing of tetracycline and GFP expression could happen in a test tube. However, without encapsulation there is no control over what is entering the system, and the reaction can degrade quickly.

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

Yes, because engineered bacteria with a tetracycline-responsive promoter that drives GFP could do this.

1.4 Describe the desired outcome of your synthetic cell operation.

At normal or zero tetracycline levels, no GFP signal. When tetracycline is present above a determined threshold concentration, the synthetic cell expresses GFP and produces a fluorescent output proportional to antibiotic concentration.

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

2.1 What would be the membrane made of?

Mainly phospholipids and cholesterol, as these form stable lipid vesicles that mimic natural cell membranes.

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

Inside, there would be a DNA circuit, which would contain the GFP gene under the control of a tetracycline-responsive promoter, bacterial cell-free Tx/Tl machinery (so ribosomes, RNA polymerase, tRNAs, amino acids, and energy regeneration machinery), and a membrane pore gene (such as α-hemolysin) to allow tetracycline to enter the vesicle of the cell.

2.3 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)

Bacterial system is ok, since tetracycline-responsive promoters work well in prokaryotic systems (with the additional benefit that this also keeps the system simpler and cheaper). No mammalian system is needed here since there is no need of a mammalian transcription factor system.

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

Tetracycline is kinda membrane-permeable, but to ensure its entry, you can induce expression of α-hemolysin (αHL) pores in the membrane. These pores allow small molecules (such as tetracycline) to enter the vesicle, triggering the genetic circuit inside.

3. Experimental details

3.1 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: phospholipids and cholesterol (for vesicle membrane)
  • Tx/Tl system: E. coli cell-free extract
  • Gene 1: aHL (alpha-hemolysin), which is the membrane pore to allow tetracycline entry
  • Gene 2: gfp (GFP reporter) under a tetracycline-responsive promoter

3.2 How will you measure the function of your system?

By using a plate reader or a fluorescence microscope, you can measure the GFP fluorescence as the tetracycline concentration is varied. After collecting this data, you can create a curve of fluorescence vs. tetracycline concentration to validate that the system activates at the right threshold. As a control, you can run the vesicles without αHL pores to confirm that tetracycline entry through the pore is required.

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 reusable biosensor strip embedded with freeze-dried cell-free reactions that coastal fishermen can dip into seawater to detect mercury contamination in their fishing grounds, producing a visible color change as a signal.

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

The strip is a fabric-like material covered with freeze-dried cell-free reactions. When a fisherman dips the strip into seawater, the water rehydrates the reaction and activates a DNA circuit responsive to free mercury ions (Hg²⁺) in the ocean. At safe mercury levels, no color change occurs. Above a threshold concentration linked to seafood safety guidelines, the circuit drives expression of a chromoprotein, producing a visible color. The strips can be manufactured cheaply, packaged dry, and distributed to fishing communities along the Colombian Pacific and Caribbean coasts, where mercury contamination from illegal gold mining that happens upstream is a documented and growing problem (Palacios-Torres et al., 2018; Marrugo-Negrete et al., 2008). Farmers near rivers could use a similar strip design to test irrigation water for heavy metal contamination before it enters their crops (Marrugo-Negrete et al., 2017). This idea doesn’t require lab equipment, electricity, training or special skills.

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

Illegal gold mining in Colombia is becoming a pressing problem. It releases large amounts of mercury and other heavy metals into rivers and coastal waters, contaminating important water ecosystems and soils that are not only home for hundreds of key species, but are also a source of food for the Colombian population. Fishing communities in particular in regions like the Chocó, the Gulf of Morrosquillo, and the Magdalena River basin rely on these waters for both food and income, but they have no practical way to know whether the fish they catch or the water they use is contaminated (Palacios-Torres et al., 2018; Marrugo-Negrete et al., 2008). According to the World Health Organization, mercury is one of the ten most hazardous substances in the world, with the Agency for Toxic Substances and Disease Registry ranking it third. It causes serious neurological damage, especially in children; different types of cancer; endothelial dysfunction; gastric and vascular disorders; liver, kidney, and brain damage; hormonal imbalances, miscarriages, and reproductive disorders; skin lesions; vision damage; and even death (Charkiewicz et al., 2025). Commercial testing requires sending samples to labs outside of the country, which proves to be expensive, slow, and highly inaccessible to these rural communities. Designing this reusable biosensor strip would help put environmental monitoring directly in the hands of the people who need it most, allowing them to make informed decisions about where to fish, when to avoid certain areas, and when to alert authorities and the public. Community organizations or environmental NGOs could help distribute them with a small guide explaining a tiered color readout (light color = caution, strong color = danger) that can give semi-quantitative information without requiring any complex measuring instruments.

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

The cells will be freeze-dried, and the seawater will activate them. Seawater is saline and contains ions, posing a challenge for the design of the strip, but the cell-free reaction can be optimized and buffered within the strip to function correctly when rehydrated with environmental water samples. During the engineering process multiple calibration tests would be needed to establish reliable thresholds. Another challenge would be storage. The strips would be freeze-dried with trehalose, a sugar derived substance that has been widely used to stabilize proteins, mammalian cells and other cell-free systems (Olsso et al., 2016). Using trehalose and sealing them individually will allow the strips to be stored at room temperature for longer (hopefully months) to allow a wider window for shipping and the use itself when needed by the communities. The best case scenario is creating a reusable strip, and testing how many times one strip can be used without losing accuracy in the results; but another challenge this proposal poses is to make sure that all the materials used for the strip are not one-use and/or disposable, creating more trash.

References:

  • Palacios-Torres, Y., Caballero-Gallardo, K., & Olivero-Verbel, J. (2018). Mercury pollution by gold mining in a global biodiversity hotspot, the Chocó biogeographic region, Colombia. Chemosphere, 193, 421-430.
  • Marrugo-Negrete, J., Benitez, L. N., & Olivero-Verbel, J. (2008). Distribution of mercury in several environmental compartments in an aquatic ecosystem impacted by gold mining in northern Colombia. Archives of Environmental Contamination and Toxicology, 55(2), 305-316.
  • Marrugo-Negrete, J., Pinedo-Hernández, J., & Díez, S. (2017). Assessment of heavy metal pollution, spatial distribution and origin in agricultural soils along the Sinú River Basin, Colombia. Environmental research, 154, 380-388.
  • Charkiewicz, A. E., Omeljaniuk, W. J., Garley, M., & Nikliński, J. (2025). Mercury exposure and health effects: what do we really know?. International journal of molecular sciences, 26(5), 2326.
  • Olsson, C., Jansson, H., & Swenson, J. (2016). The role of trehalose for the stabilization of proteins. The Journal of Physical Chemistry B, 120(20), 4723-4731.

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)

Astronauts (especially the ones that go on long-duration missions) face difficult psychological challenges: communication delays with Earth, profound feelings of isolation, disrupted circadian rhythms from irregular light exposure in orbit, poor sleep and the worsening of cognitive performance (Collins, 2003). Helping them regulate their circadian rhythms could be an important step for sleep mediation, stress resilience and mental peace during their missions. Melatonin is the hormone whose synthesis depends on enzymatic pathways sensitive to light cues. Understanding how melatonin works inside astronauts’ bodies could be key to protecting astronaut mental health.

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)

The enzyme AANAT (arylalkylamine N-acetyltransferase) is the rate-limiting enzyme in melatonin biosynthesis. Its upstream regulator is the CLOCK gene circadian transcription factor.

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

AANAT enzymatic activity controls the nightly increase in melatonin that drives circadian rhythm synchronization. In space, the absence of normal 24-hour light/dark cycles suppresses AANAT expression, reducing melatonin production and fragmenting sleep (Zong et al., 2025). Using BioBits to express AANAT in space under simulated circadian promoter control can be an opportunity to test whether the cell-free Tx/Tl machinery can reliably produce the AANAT enzyme on demand. If results are positive, astronauts could use a biosynthesis system that supplements melatonin during their mission, avoiding pre-packed pharmaceuticals that can degrade over long space missions.

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

Hypothesis: BioBits cell-free reactions aboard spacecraft will produce equal or slightly lower yields of AANAT protein in comparison to Earth, creating a successful biosynthesis system that supplies astronauts with melatonin during space travel.

Melatonin deficiency is already documented in astronauts, but it is unclear how much of this is due to light exposure versus a genuine suppression of the biosynthetic machinery itself (Zong et al., 2025). If cell-free reactions can produce AANAT in space at comparable yields to Earth, this validates the concept of on-demand biosynthesis of melatonin (and perhaps other psychoactive molecules) aboard spacecraft. If yields are lower, it points to key specific technical challenges (for example: DNA/mRNA damage due to space radiation) that could be solved before in-space biomanufacturing of therapeutics becomes a reality.

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) First, create freeze-dried BioBits pellets that contain the AANAT gene attached to an sfGFP reporter, to allow fluorescence to signal if protein expression is successful. One set of pellets will be activated aboard the spacecraft, while an identical set is activated on Earth as the first control. The two additional controls are: (1) pellets with an always-on gene (positive control), and (2) pellets with no DNA (negative control). Astronauts will rehydrate one pellet every four hours over 24 hours to mimic a circadian cycle. Protein expression will be analyzed and measured using the fluorescence viewer for real-time results. Higher brightness means more AANAT protein was made.

References:

  • Collins, D. L. (2003). Psychological issues relevant to astronaut selection for long-duration space flight: a review of the literature. Journal of Human Performance in Extreme Environments, 7(1), 1.

  • Zong, H., Fei, Y., & Liu, N. (2025). Circadian disruption and sleep disorders in astronauts: a review of multi-disciplinary interventions for long-duration space missions. International Journal of Molecular Sciences, 26(11), 5179.