Week 9 HW: Cell-Free Systems

My Homework

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.

Cell-free protein synthesis offers significant advantages over in vivo systems due to its flexibility and precise control over experimental conditions. Components can be easily added, removed, or adjusted without affecting cell viability, enabling rapid optimization and high-throughput experimentation. This system allows incorporation of non-natural amino acids to create proteins with novel properties, which is difficult in living cells. It is also ideal for producing toxic proteins that would otherwise harm cells. Additionally, cell-free systems are fast, scalable, reproducible, and suitable for prototyping genetic circuits and biosensors, including portable and freeze-dried applications for field use.

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

The following table was made based on the information provided in Cui., et al (2022).

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 and regeneration are critical in cell-free systems because many biochemical processes, especially protein synthesis, are highly ATP-dependent. ATP is continuously consumed during transcription, translation, and enzymatic reactions. Without regeneration, ATP would be rapidly depleted, leading to early termination of the reaction and low product yield. Additionally, supplying ATP in stoichiometric amounts is impractical due to its high cost, making regeneration systems essential for sustained and scalable cell-free reactions. In the system described by Yadav et al., (2025) ATP is regenerated from pyruvate through a two-step enzymatic pathway. First, the enzyme pyruvate oxidase (Pox5) converts pyruvate + inorganic phosphate (Pi) + oxygen into acetyl phosphate, a high-energy intermediate. Then, acetate kinase (AckA) transfers the phosphate group from acetyl phosphate to ADP, producing ATP and acetate. This allows ATP to be regenerated in situ during the reaction using pyruvate as the energy source. An additional enzyme, catalase (KatE), is required because the pyruvate oxidase reaction generates hydrogen peroxide (H₂O₂) as a byproduct. Catalase breaks down H₂O₂ into water and oxygen, preventing damage to proteins and maintaining system activity . This ATP regeneration system was successfully tested in the PURE cell-free system, where it supported protein synthesis. Importantly, when this pyruvate-based system was combined with the traditional creatine phosphate/creatine kinase system, the highest protein yield was achieved (up to ~230 μg/mL), outperforming either system alone.

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

Prokaryotic and eukaryotic cell-free expression (CFE) systems differ mainly in cost, speed, and protein complexity. Prokaryotic systems (e.g., E. coli) are widely used because they are fast, inexpensive, and produce high protein yields, making them ideal for simple proteins. However, they often lack chaperones and post-translational modification (PTM) machinery, which limits proper folding and functionality of complex eukaryotic proteins. In contrast, eukaryotic systems (e.g. rabbit reticulocyte extracts) are pricier and slower to prepare, but they can correctly express proteins requiring PTMs such as disulfide bond formation, glycosylation, and membrane protein insertion, which are essential for many functional proteins. For a prokaryotic system, a suitable protein is amilCP, a chromoprotein reporter that is simple, does not require PTMs, and can be easily produced. In contrast, the Buntru et al. (2014) study demonstrates that eukaryotic systems such as cell-free systems from Nicotiana tabacum are ideal for complex proteins such as full-length antibodies, which require correct folding, disulfide bond formation, and assembly of heavy and light chains; their system successfully produced functional antibodies. Another example from the same study is glucose oxidase (GOx), a glycosylated enzyme whose activity depends on proper folding and glycosylation, both achieved in the eukaryotic cell-free system.

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.

To optimize the expression of a membrane protein in a cell-free system, I would design the experiment by combining a CFPS (cell-free protein synthesis) system with lipid vesicles (like liposomes). First, I would set up a basic CFPS reaction with: DNA encoding the membrane protein Cell extract, amino acids, energy system Then, I would add liposomes or microsomes to the reaction so that the membrane protein can insert directly into a lipid bilayer during synthesis. This is important because membrane proteins need a membrane to fold correctly . Next, I would optimize key conditions, such as: Temperature and pH Magnesium and salt concentrations DNA concentration Lipid composition of vesicles The main challenges in expressing membrane proteins in cell-free systems include misfolding, low yield, improper membrane insertion, and lack of post-translational modifications. These problems occur because membrane proteins are hydrophobic and structurally complex, making them unstable in aqueous conditions and difficult to produce efficiently. To address this, lipid vesicles such as liposomes or microsomes are added to provide a membrane-like environment for proper folding and insertion, and reaction conditions (such as temperature, ions, and DNA concentration) are optimized to improve yield (Takeda, et al., 2015; Mayeux, et al., 2021; Kim, et al., 2025). Additionally, chaperones or cofactors can be included to assist folding. Importantly, the choice of system depends on the protein: prokaryotic cell-free systems (e.g., E. coli lysate + liposomes) are suitable for simpler membrane proteins, while eukaryotic systems (with microsomes) are better for complex proteins that require proper folding and post-translational modifications, such as receptors or GPCRs (Takeda, et al., 2015).

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.

First, RNase contamination can degrade mRNA and reduce protein production. This often comes from plasmid preparation kits. To fix this, RNase inhibitors should be added to the reaction (e.g., Murine RNase Inhibitor), and clean, nuclease-free reagents should be used . Second, problems with the template DNA design and contamination can reduce translation efficiency. For example, incorrect regulatory elements, poor ribosome binding sites, or secondary structures at the 5′ end can interfere with translation initiation. To solve this, the DNA sequence should be verified and optimized, for example by modifying the 5′ region to eliminate secondary structures or improving the initiation sequence. Residual amounts of SDS, ethidium bromide and ammonium acetate often found in plasmid preparation and PCR product gel purification can inhibit translation. Third, non-optimal template DNA concentration can affect yield. Too little DNA produces low mRNA levels, while too much DNA can overwhelm the translation machinery. Depending on the kit used, it is important to check the recommended amount of DNA that should be used.

Homework question from Kate Adamala

Design an example of a useful synthetic minimal cell as follows: This idea is based on Chen, et al., (2024) & Yang, et al., (2025)

1. Pick a function and describe it. a. What would your synthetic cell do? What is the input and what is the output? Detect plant pathogen-associated signals in vivo and generate a visible signal in plant tissue. Input: chitooligosaccharides (COs) or lipo-chitooligosaccharides (LCOs) from fungi/bacteria. Output: production and release of betalain pigment (RUBY system), visible as purple coloration. The RUBY system consists of three enzymes that synthesize betalain pigment, enabling direct visualization.

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

No. Encapsulation is required to incorporate membrane receptors and maintain compartmentalization for signal detection and controlled output.

c. Could this function be realized by genetically modified natural cell?

Yes, plants can express RUBY reporters. However, synthetic cells provide a non-living, deployable biosensor without genetic modification of the plant.

d. Describe the desired outcome of your synthetic cell operation.

In the presence of pathogen-derived molecules, the synthetic cell produces and releases betalain pigment, generating a visible purple signal in infected plant tissue.

2. Design all components that would need to be part of your synthetic cell. a. What would be the membrane made of? Phospholipids (POPC) + cholesterol.

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

Cell-free Tx/Tl system (E. coli-based), RUBY genes (CYP76AD1, DODA, Glu-T), Energy mix (ATP, amino acids), Synthetic signal transduction module. The signal transduction module consists of a membrane-associated sensing system inspired by LysM receptor-like kinases that recognizes chitooligosaccharides (COs) or lipo-chitooligosaccharides (LCOs) outside the synthetic cell. Because full-length LysM receptors are complex eukaryotic transmembrane proteins that are difficult to express and fold correctly in an E. coli-based cell-free system, this design uses a simplified or engineered receptor module compatible with bacterial Tx/Tl. Upon ligand binding, the sensor undergoes a conformational change that activates a synthetic intracellular transcriptional regulator, which in turn induces expression of the RUBY genes. This coupling allows external pathogen-derived signals to be converted into internal gene expression, resulting in production of betalain pigment that can diffuse out of the synthetic cell and generate a visible signal in plant tissue.

c. 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 (E. coli-based), sufficient for enzyme production.

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

Detection occurs through a membrane-associated sensing module inspired by LysM receptors, which bind external COs/LCOs. The signal is transduced internally via a synthetic transcriptional activator, inducing RUBY expression. The betalain pigment diffuses or exits through membrane pores (e.g., α-hemolysin) into plant tissue.

3. Experimental details a. 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, cholesterol Genes: RUBY system: CYP76AD1, DODA, Glu-T Synthetic sensing module (LysM-inspired) α-hemolysin (optional pore)

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

Visual detection of purple pigment in plant tissue and quantification using imaging or spectrophotometry.

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. This idea is inspired by Rohde, Niehl & Ziebell (2025). I propose a wearable and attachable biosensing system using freeze-dried cell-free reactions to detect plant viruses like Tomato brown rugose fruit virus (ToBRFV) on clothing, tools, and agricultural surfaces through a visible color change.

2. How will the idea work, in more detail? Write 3-4 sentences or more. Freeze-dried cell-free biosensors would be integrated into wearable fabrics (gloves, clothes, sleeves) and also into attachable patches or stickers that can be placed on tools (scissors, knives, trays) or greenhouse surfaces. These systems would contain RNA-sensitive modules (e.g., toehold switches or CRISPR-based detection) designed to recognize ToBRFV genomic RNA. When viral particles from contaminated plant sap contact the material and provide moisture, the system activates and produces a visible color signal. This allows real-time detection of contamination across multiple points of contact, helping growers identify and limit virus spread during routine activities.

3. What societal challenge or market need will this address? ToBRFV spreads easily through mechanical transmission and can persist on hands, clothing, and tools, making outbreaks difficult to control. Current diagnostics are lab-based and do not monitor contamination during daily operations. This system addresses the need for rapid, on-site contamination detection across the entire workflow, improving hygiene practices, reducing crop losses, and enabling better disease management in greenhouse and field production.

4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? Activation with water: Sensors activate upon contact with plant sap, humidity, or by applying a simple spray buffer during inspections. Stability: Freeze-dried reactions are stabilized within protective coatings or hydrogels embedded in fabrics or patches. One-time use: Use replaceable biosensor patches that can be attached to clothing, tools, or surfaces and discarded after activation. Versatility across materials: Design sensors as modular stickers or strips that can be applied to different surfaces, not just textiles. This approach turns everyday agricultural materials into active biosensing surfaces, enabling continuous monitoring of pathogens spread across the entire production system.

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)

Space environments expose organisms to microgravity and increased radiation, which can alter microbial survival, gene expression, and evolution. Understanding how extremophiles respond to these conditions is important for astronaut health, planetary protection, and long-term space missions. Microbes that survive harsh Earth environments (e.g., deserts, Antarctic systems) are ideal models to study resilience in space. Cell-free systems provide a safe and low-resource method to analyze genetic responses without maintaining living cultures, making them well-suited for space biology experiments.

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)

Stress-response genes (DNA repair, radiation resistance) and conserved microbial markers (16S rRNA).

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

Stress-response genes are directly involved in microbial survival under radiation and microgravity conditions. By monitoring these genes, we can assess how microbes adapt to space environments. Conserved genes such as 16S rRNA confirm microbial presence, while functional genes provide insight into biological responses to stress. This allows us to understand not only whether microbes survive in space, but how their molecular mechanisms change under these conditions.

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

We hypothesize that microbial DNA exposed to space conditions will show detectable differences in stress-response gene signatures that can be measured using a BioBits® cell-free system. If DNA from microbes exposed to radiation or simulated microgravity is amplified using miniPCR, the cell-free system can detect specific gene targets and produce a measurable fluorescent signal. By comparing samples exposed to space-like conditions with ground controls, we can identify changes in gene abundance or presence related to stress adaptation. The goal is to develop a portable system to monitor microbial responses in space, contributing to understanding microbial survival, evolution, and potential risks during long-duration missions.

7. 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)

DNA samples from microbes exposed to simulated space conditions (radiation or microgravity analogs) and control samples will be tested. Target genes will be amplified using miniPCR. Amplified DNA will be added to BioBits® reactions programmed to detect specific sequences. Fluorescence will be measured using the P51 viewer. Differences in signal between exposed and control samples will indicate changes in stress-response gene detection, allowing assessment of microbial adaptation to space conditions.

References

Buntru, M., Vogel, S., Stoff, K., Spiegel, H., & Schillberg, S. (2015). A versatile coupled cell‐free transcription–translation system based on tobacco by‐2 Cell lysates. Biotechnology and Bioengineering, 112(5), 867–878. https://doi.org/10.1002/bit.25502

Chen, L., Cai, Y., Liu, X., Yao, W., Wu, S., & Hou, W. (2024). The ruby reporter for visual selection in soybean genome editing. aBIOTECH, 5(2), 209–213. https://doi.org/10.1007/s42994-024-00148-6

Cui, Y., Chen, X., Wang, Z., & Lu, Y. (2022). Cell-free pure system: Evolution and achievements. BioDesign Research, 2022, 9847014. https://doi.org/10.34133/2022/9847014

Kim, W., Han, J., Chauhan, S., & Lee, J. W. (2025). Cell-free protein synthesis and vesicle systems for programmable therapeutic manufacturing and delivery. Journal of Biological Engineering, 19(1). https://doi.org/10.1186/s13036-025-00523-x

Mayeux, G., Gayet, L., Liguori, L., Odier, M., Martin, D. K., Cortès, S., Schaack, B., & Lenormand, J.-L. (2021). Cell-free expression of the outer membrane protein oprf of pseudomonas aeruginosa for vaccine purposes. Life Science Alliance, 4(6). https://doi.org/10.26508/lsa.202000958

Rohde, M. J., Niehl, A., & Ziebell, H. (2025). A novel tobrfv cdna full-length infectious clone provides insights on virus-host range and inoculation strategies. Plant Disease, 109(10), 2123–2134. https://doi.org/10.1094/pdis-08-24-1665-re

Takeda, H., Ogasawara, T., Ozawa, T., Muraguchi, A., Jih, P.-J., Morishita, R., Uchigashima, M., Watanabe, M., Fujimoto, T., Iwasaki, T., Endo, Y., & Sawasaki, T. (2015). Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Scientific Reports, 5(1). https://doi.org/10.1038/srep11333

Yadav, S., Perkins, A. J., Liyanagedera, S. B., Bougas, A., & Laohakunakorn, N. (2025). ATP regeneration from pyruvate in the pure system. ACS Synthetic Biology, 14(1), 247–256. https://doi.org/10.1021/acssynbio.4c00697

Yang, X., Tannous, J., Rush, T. A., Del Valle, I., Xiao, S., Maharjan, B., Liu, Y., Weston, D. J., De, K., Tschaplinski, T. J., Lee, J. H., Morgan, M., Jacobson, D., Islam, M. T., Chen, F., Abraham, P. E., Tuskan, G. A., Doktycz, M. J., & Chen, J.-G. (2025). Utilizing plant synthetic biology to accelerate plant-microbe interactions research. BioDesign Research, 7(2), 100007. https://doi.org/10.1016/j.bidere.2025.100007