Week 9 — 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. Cell-free protein synthesis offers greater flexibility and control compared to in vivo systems because it allows precise manipulation of reaction conditions such as component concentrations, temperature, and reaction time. Additionally, it eliminates cellular interference, such as metabolic regulation, toxicity effects, and competing pathways, enabling more efficient and tunable protein production. One case where cell-free expression is advantageous is in the production of toxic proteins, such as toxins or antimicrobial peptides, which would otherwise damage or kill the host cell. Another case is the synthesis of proteins requiring non-natural amino acids or specialized conditions, which are difficult to achieve in living cells due to their tightly regulated environment.

2. Describe the main components of a cell-free expression system and explain the role of each component. A cell-free expression system is composed of several essential components that enable protein synthesis outside of living cells. First, it includes a genetic template (DNA or mRNA) that contains the gene of interest. If DNA is used, transcription machinery such as RNA polymerase is required to synthesize mRNA. The system also contains the translation machinery, including ribosomes, tRNAs, amino acids, and translation factors, which work together to synthesize the protein. Additionally, an energy regeneration system is required, supplying molecules such as ATP, GTP, and other energy substrates to sustain the reaction. Finally, buffers and salts are included to maintain optimal physicochemical conditions, such as pH and ionic strength, which are necessary for proper enzyme activity and protein synthesis.

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 protein synthesis is a highly energy-intensive process that consumes ATP and GTP during transcription and translation. Since cell-free systems lack metabolic pathways to recycle energy, ATP is rapidly depleted, leading to an early توقف of protein production. Therefore, continuous energy regeneration is necessary to sustain the reaction and achieve higher protein yields. One common method to ensure continuous ATP supply is the use of an energy regeneration system based on phosphoenolpyruvate (PEP), which acts as a high-energy phosphate donor to regenerate ATP from ADP, maintaining the reaction over time.

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 systems differ mainly in their complexity and ability to perform post-translational modifications. Prokaryotic systems, such as those derived from E. coli, are simpler, faster, and more cost-effective, but they lack the machinery for most post-translational modifications. In contrast, eukaryotic systems are more complex and can perform modifications such as glycosylation and proper protein folding. A suitable protein for production in a prokaryotic system is Green Fluorescent Protein (GFP), as it is relatively simple and does not require post-translational modifications. On the other hand, monoclonal antibodies are better produced in eukaryotic systems because they require correct folding, disulfide bond formation, and glycosylation to be functional.

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 an experiment that combines a suitable cell-free extract with a membrane-mimicking environment, because membrane proteins tend to aggregate or misfold in aqueous solution if their hydrophobic regions are not stabilized during synthesis. It would begin with an E. coli extract for simple bacterial membrane proteins, or a eukaryotic/microsome-containing system for more complex proteins requiring eukaryotic folding or post-translational processing. Then I would test additives such as mild detergents, liposomes, or nanodiscs to promote co-translational insertion and stabilize the protein in a native-like environment. The main challenges here are low solubility, aggregation, incorrect folding, and loss of activity. To address these, It would be good to run a small optimization screen varying temperature, Mg²⁺/K⁺ concentrations, DNA template amount, incubation time, and the type and concentration of membrane mimic. Lowering the temperatures can reduce aggregation, while liposomes or nanodiscs often improve folding and functionality better than detergents alone. If yield is low, I would use a continuous-exchange cell-free format to extend reaction time and improve protein production. To evaluate success, I would not only measure total protein yield, but also test whether the membrane protein is soluble, properly inserted, and functional. This could be done using SDS-PAGE for expression, centrifugation to compare soluble versus insoluble fractions, and activity or ligand-binding assays depending on the protein. In this way, the experiment would optimize both expression and functional quality, not just the amount of protein produced.

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. Low yield in a cell-free system can result from several factors. First, the DNA template may be poor or suboptimal, for example if it is degraded, contains inhibitors, or has weak regulatory elements, which reduces transcription and translation efficiency. A good troubleshooting strategy would be to verify template quality, adjust DNA concentration, and include a positive control to confirm that the system itself is working properly. Second, the reaction conditions may not be optimal, such as incorrect Mg²⁺ or K⁺ concentrations, unsuitable temperature, or an incubation time that is too short. These variables strongly affect ribosome activity and protein synthesis. To troubleshoot this, I would run a small optimization screen varying salt concentrations, temperature, and reaction time to identify the best expression conditions. Third, the target protein itself may be unstable, misfolded, or prone to aggregation, especially if it is a difficult protein such as a membrane or disulfide-bonded protein. In that case, even if it is synthesized, it may not accumulate properly. A good strategy would be to add folding-supportive components such as detergents, liposomes, nanodiscs, redox helpers, or chaperone-like additives depending on the protein type.

Homework question from Kate Adamala

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

1. Pick a function and describe it. a. What would your synthetic cell do? What is the input and what is the output? b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation? c. Could this function be realized by genetically modified natural cell? d. Describe the desired outcome of your synthetic cell operation.

2. Design all components that would need to be part of your synthetic cell. e. What would be the membrane made of? f. What would you encapsulate inside? Enzymes, small molecules. g. 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) h. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

3. Experimental details i. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.) j. How will you measure the function of your system?

A useful synthetic minimal cell could be designed to detect Salmonella enterica contamination on food and release bacteriophages only when the pathogen is present. The function of this system is targeted antimicrobial delivery. The input would be a Salmonella-associated quorum-sensing molecule (AI-2), and the output would be the release of lytic anti-Salmonella enterica phages. This function cannot be fully realized by cell-free Tx/Tl alone without encapsulation, because the system requires physical containment and controlled release of phage particles. However, a genetically modified natural cell could potentially perform a similar sensing-response function, although it would be less controllable and may raise biosafety and regulatory concerns, especially in food applications. The desired outcome of this synthetic cell is to improve food safety by reducing Salmonella contamination, while avoiding unnecessary phage release in clean food, making the system more efficient and cost-effective. The synthetic cell would be based on a liposome membrane composed of phospholipids and cholesterol, providing structural stability. Inside the liposome, lytic anti-Salmonella enterica phages and an inactive phospholipase A2-type enzyme would be encapsulated. The membrane would display the LsrB protein, which binds the quorum-sensing molecule AI-2. When AI-2 is detected, binding to LsrB would trigger activation of the phospholipase, which then destabilizes the membrane and causes the release of phages. This design does not require an internal Tx/Tl system, since the phages are pre-formed and only need to be released. If needed, a bacterial system would be sufficient, as no complex post-translational modifications are required. The synthetic cell communicates with the environment through surface-level detection, meaning the signal (AI-2) does not need to enter the cell but only bind to the membrane protein. The main components include phospholipids, cholesterol, the lsrB gene (for the binding protein), and a gene encoding a phospholipase enzyme. To evaluate the system, its function can be measured by quantifying the reduction of Salmonella in treated food samples using qPCR, comparing results with untreated controls. A successful system would show a significant decrease in Salmonella only when the trigger molecule is present.

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 childrens beanie incorporating freeze-dried cell-free systems that detect high UV exposure, trigger a cooling hydrogel response, and send alerts to caregivers to prevent heat stress and sun overexposure.

- How will the idea work, in more detail? Write 3-4 sentences or more. The beanie contains freeze-dried cell-free sensor modules embedded within the fabric that are activated by moisture from sweat or environmental humidity. These systems are designed to detect high levels of UV radiation using UV-responsive genetic elements. When a threshold level of exposure is reached, the system triggers two responses: activation of a compartment containing a cooling hydrogel that expands or releases stored moisture to reduce temperature, and generation of a signal that can be detected by a small integrated electronic module, which sends a notification to a caregiver phone. This allows real-time monitoring and immediate response to potentially harmful exposure conditions.

- What societal challenge or market need will this address? This system addresses the risk of heat stress and excessive sun exposure in children, who are more vulnerable to dehydration and sun damage and may not recognize early warning signs. It provides a preventive, wearable solution for parents and caregivers, especially in outdoor settings such as parks, schools, and sports activities. The product also aligns with increasing demand for smart textiles and health-monitoring wearables.

- How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? One limitation of cell-free systems is that they require activation, which in this case is solved by using sweat and moisture as natural triggers during wear. Stability is addressed through freeze-drying (lyophilization), allowing long-term storage within the fabric until activation. Since cell-free reactions are typically single-use, the beanie could be designed with replaceable or modular sensing patches that can be swapped after activation. Additionally, integrating the system into protected compartments within the textile helps maintain functionality and prevents environmental degradation.

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) Biofilms are a major concern in spacecraft because they can help bacteria persist on cabin surfaces, resist cleaning, and potentially threaten astronaut health and equipment. In closed environments such as space habitats, microbes may respond differently to stressors like radiation and microgravity. Understanding whether space-like conditions increase the biofilm potential of common surface bacteria is important for long-term missions. This topic is significant for humanity because safer microbial control will be essential as people spend longer periods in space, and it is scientifically interesting because bacterial adaptation in space is still not fully understood.

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 molecular target is the icaA gene in Staphylococcus epidermidis, a biofilm-associated gene involved in polysaccharide matrix production and surface colonization.

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) The icaA gene is directly related to biofilm formation, which allows bacteria to attach to surfaces and persist under harsh conditions. In a spacecraft cabin, this is especially relevant because bacteria growing as biofilms may be harder to remove and more resistant to cleaning procedures. By focusing on icaA, this project investigates whether space-like stress conditions are associated with stronger biofilm-related adaptation in Staphylococcus epidermidis. Detecting this gene under simulated space conditions would help us understand whether bacteria in spacecraft environments may become more persistent and create greater risks for both crew health and spacecraft materials.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) My hypothesis is that Staphylococcus epidermidis exposed to space-like stress conditions, specifically microgravity-like conditions and radiation stress, will show a stronger biofilm-associated genetic signature related to icaA than bacteria grown under normal Earth conditions. The reasoning is that bacteria in stressful environments often adapt to improve survival, and biofilm formation is one of the main strategies used to resist environmental stress. If space-like conditions favor this adaptation, then bacteria commonly found on spacecraft surfaces could become more persistent and harder to eliminate. The goal of this project is to test whether simulated space stress changes the detectability of icaA using a compact molecular workflow that combines miniPCR, the BioBits cell-free system, and fluorescence readout. This could help identify biofilm risk early during future space missions.

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) I would culture Staphylococcus epidermidis under two conditions: normal Earth conditions and simulated space-like stress conditions including microgravity-like growth and radiation exposure. DNA would be extracted from both groups, and icaA would be amplified using miniPCR. The amplified products would then be linked to a BioBits cell-free fluorescent reporter reaction and visualized with the P51 Molecular Fluorescence Viewer. Controls would include a no-template control and a known icaA-positive control. Data collected would include PCR amplification success, fluorescence intensity, and comparison of signal strength between stressed and unstressed samples.