Week 9 homework

Cell-free systems 🧪

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.

Compared to conventional in vivo methods, cell-free protein synthesis provides modularity and substantially higher experimental control, as all the system’s components can be readily added or removed, especially when the strategy employed is to separately produce or extract each cellular element required for the process and then combine them all together into a single reaction. Cell-free systems also offer the potential for precise control over reaction conditions, such as pH and ion concentration, while being more flexible and versatile since they allow the expression of proteins deleterious to living cells, support the integration of non-natural and non-canonical amino acids into peptide backbones, and are compatible with diverse DNA templates (linear or plasmid). Additionally, they eliminate constraints imposed by the existence of living cells. For instance, unlike traditional cell cultures, they do not need any monitoring, cultivating, or other interventions aimed at preservation, nor are they susceptible to issues of cell viability, growth limits, or stress responses. Similarly, since the cell-free apparatus exists outside of the context of a cellular platform, there are no cell-membrane barriers, facilitating access to biochemical reactions, while, at the same time, there is no interference or competition from other metabolic procedures or regulatory signals, enabling all the available resources to be channeled towards the synthesis of the desired protein, which, in addition, can later be purified more easily, without impurities. The absence of living cells can be translated into abolishing the need for cloning and cellular transformation as well, which, in turn, ensures safer handling, as no genetically modified organisms are involved in cell-free protein production. More generally, one of the method’s most significant advantages is that it is a highly efficient technique for rapid protein synthesis that can also withstand being transferred across larger distances for longer periods of time, as the entire system can be easily freeze-dried and stored for later use ¹.

For more tangible examples, more specific cases where cell-free expression is more beneficial than cell-dependent protein production are presented below:

• In theranostic applications, where the system has to be implanted in close proximity or inside the human body. Since no living cells are implicated, whose parts could potentially be recognized as harmful agents, the probability for a toxic immune or allergic reaction is low.
• In experiments conducted to study the foundations of transcription and translation. The isolation of a cell-free platform ensures the appropriate conditions to investigate gene expression mechanisms without the background noise from other cellular processes.
• For remote field testing, as cell-free systems generally require far less infrastructure than traditional cell-based production installations. Because of this, cell-free platforms can very easily be converted into portable platforms, enabling carrying out experiments, for instance, even in space.
• For on-demand biomanufacturing, since, not only are all the system’s resources directed to the generation of the desired product, but also cell-free systems can achieve higher titers in considerably less time (minutes to hours instead of days). Apart from the efficiency, the desired product is less contaminated with unwanted cellular metabolites, allowing for higher purity and, therefore, for the implementation of less complex purification methods.

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

The main input of cell-free systems is a circular or linear DNA sequence that contains the gene to be expressed (including an appropriate promoter), while the principal output is a desired protein. The first step for the expression of the gene involves its transcription, for which the enzyme RNA polymerase is required, along with Mg²⁺ ions, which act as essential co-factors. For the mRNA of the desired gene to be physically synthesized, the cell-free system should contain the needed building blocks too, namely nucleotides. To effectively translate the transcript into protein, the reaction should also have access to ribosomes and tRNA molecules, which will build the peptide sequence and carry the amino acids (found in the solution too) to the correct position of the nascent peptide respectively. Lastly, the energy required for this entire machinery to function can be obtained with the addition of ATP into the cell-free system.

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.

Despite their many advantages, cell-free systems are also characterized by their inability to regenerate energy, mostly due to their lack of intricate cellular structures, such as intracellular compartments and membrane protein complexes, which in naturally occurring cellular systems, play a major role in energy-procuring metabolic pathways, such as glycolysis and the Krebs Cycle. For this reason, it is critical to “recharge” cell-free platforms of protein production with the frequent addition of ATP.

However, instead of constantly supplying a cell-free system with external ATP, it is more efficient to embed the capability of regenerating its energy resources into the cell-free reaction from the beginning. To achieve this, the most straightforward way involves adding two more factors to the components listed in the previous question, which can restore ATP from ADP (the product of ATP expenditure). Those two reagents are, firstly, a molecule that acts as a donor of a phosphate group and, secondly, an enzyme, a kinase in particular, that can catalyze the “reattachement” of the phosphate moiety to ADP to produce ATP in what is biochemically called a “substrate-level phosphorylation”. Fortunately, there are multiple combinations of such reagents that can readily be used in cell-free systems, with the most popular including creatine phosphate and creatine kinase ², acetyl phosphate and acetate kinase ^{3 4}, as well as polyphosphate and polyphosphate kinase ².

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, most commonly derived from Escherichia coli extracts, are valued for their speed, low cost, and high protein yield. These systems contain the transcriptional and translational machinery needed to synthesize proteins in vitro without living cells, making them ideal for rapid protein production and synthetic biology applications. However, they have limited ability to perform complex post-translational modifications such as glycosylation or correct disulfide bond formation. A suitable protein to produce in a prokaryotic cell-free system would be insulin or green fluorescent protein (GFP). GFP is especially appropriate because it is relatively small, folds efficiently in bacterial conditions, and does not require extensive modifications to become functional, allowing high yields and straightforward analysis.

Eukaryotic cell-free expression systems, derived from sources such as wheat germ, insect cells, or rabbit reticulocytes, are more suitable for synthesizing complex eukaryotic proteins. Although these systems are generally more expensive and slower than prokaryotic systems, they better support proper protein folding, disulfide bond formation, and post-translational modifications. A good example of a protein to produce in a eukaryotic cell-free system is a monoclonal antibody or a membrane receptor such as the human epidermal growth factor receptor (EGFR). These proteins require sophisticated folding and modification machinery to function correctly, which eukaryotic systems can provide. Therefore, the choice between prokaryotic and eukaryotic cell-free expression depends largely on the structural complexity and modification requirements of the target protein.

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.

Designing a cell-free experiment to optimize membrane protein expression would involve systematically testing reaction conditions that improve protein synthesis, folding, and membrane insertion. I would use a eukaryotic cell-free system, such as a wheat germ or insect-cell extract, because membrane proteins often require complex folding machinery and a lipid environment. The experiment would include adding artificial membrane mimics such as liposomes, nanodiscs, or detergent micelles directly into the reaction mixture so the newly synthesized protein can insert into a membrane-like structure during translation. To optimize expression, I would vary factors such as magnesium and potassium ion concentrations, temperature, reaction duration, and the concentration of membrane additives. Protein yield and functionality could then be measured using fluorescence tags, Western blotting, or ligand-binding assays to identify the conditions that produce the highest amount of properly folded protein.

One major challenge in membrane protein expression is that these proteins are highly hydrophobic and tend to aggregate or misfold when removed from a membrane environment. Another difficulty is achieving correct orientation and functional conformation, since many membrane proteins rely on lipid interactions for stability. To address these issues, I would include nanodiscs or specific lipid compositions that resemble the protein’s natural membrane environment, helping stabilize the protein during synthesis. Molecular chaperones could also be added to assist folding and reduce aggregation. Because overexpression can overwhelm the system and produce inactive protein, I would test different DNA template concentrations and slower reaction temperatures to encourage proper folding. By combining controlled optimization with membrane-mimicking components and folding aids, the experiment would increase the likelihood of producing a functional membrane protein suitable for structural or biochemical studies.

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.

One possible reason for low protein yield is poor quality or degraded DNA template. If the plasmid or linear DNA used in the cell-free reaction is damaged, transcription efficiency will decrease and less mRNA will be available for translation. To troubleshoot this issue, I would verify DNA integrity using gel electrophoresis, measure purity with spectrophotometry, and prepare fresh, highly purified DNA templates. Using stronger promoters or optimizing codon usage could also improve transcription and translation efficiency.

A second cause may be inefficient transcription or translation conditions within the reaction mixture. Incorrect concentrations of magnesium ions, potassium ions, amino acids, or energy substrates can significantly reduce protein synthesis. To address this, I would systematically optimize reaction conditions by testing different ion concentrations, incubation temperatures, and reaction times. Small-scale screening experiments would help identify the combination that produces the highest protein yield.

A third issue could be rapid degradation of mRNA or the synthesized protein by nucleases or proteases present in the extract. Degradation reduces the amount of functional product that accumulates during the reaction. Troubleshooting strategies would include using nuclease- or protease-deficient extracts, adding RNase inhibitors or protease inhibitors, and minimizing reaction handling time to protect reaction components from contamination.

Another possible reason is improper protein folding, especially for large, complex, or membrane-associated proteins. Misfolded proteins may aggregate and become inactive, reducing the apparent yield of soluble target protein. To solve this problem, I would lower the reaction temperature to slow translation and improve folding, add molecular chaperones to assist proper protein assembly, or include detergents, liposomes, or nanodiscs if working with membrane proteins.

Finally, codon bias can limit protein production if the target gene contains codons that are rarely used by the expression system. In prokaryotic systems, rare codons can stall ribosomes and decrease translation efficiency. A useful troubleshooting strategy would be codon optimization of the gene sequence for the host extract being used or supplementing the reaction with rare tRNAs. This can improve ribosome movement and increase overall protein synthesis.

Homework question from Kate Adamala

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

1. Pick a function and describe it.

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

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

• What would the membrane be made of?
• What would you encapsulate inside? Enzymes, small molecules.
• Which organism will your Tx/Tl system come from? Is bacterial OK, or do you need a mammalian system for some reason?
• How will your synthetic cell communicate with the environment?

3. Experimental details

• List all lipids and genes.
• How will you measure the function of your system?

For this part of this week’s homework, I would like to design a synthetic minimal Cell for saltwater desalination, as analyzed below.

1. Proposed function of the synthetic minimal cell

The proposed synthetic minimal cell is designed to remove sodium chloride (NaCl) from saltwater through biologically inspired selective ion transport, such as ion transport naturally occurring in kidney epithelial cells, halophilic microorganisms, and marine ion-regulating organisms. In more detail, the synthetic cell would act as a programmable microscopic desalination unit that, upon being placed in saltwater (input), selectively imports sodium (Na⁺) and chloride ions (Cl^−^) from the environment and traps them internally. As a result, apart from the accumulation of NaCl inside the synthetic cells, this system would produce water with reduced salt concentration (output).

Such a function could not be realized by cell-Free Tx/Tl alone, as desalination fundamentally depends on compartmentalization and membrane gradients. Without encapsulation, ion gradients cannot be maintained, nor can selective ion accumulation or osmotic separation be achieved. Another obstacle that arises from a simple cell-Free Tx/Tl reaction is that a mechanism of ATP-driven active transport cannot be established, which, in a synthetic cell-based system, would provide the electrochemical potential needed for the removal of NaCl from the surrounding water.

On the contrary, desalination could be carried out by genetically modified natural cells, such as genetically enhanced halophilic bacteria or engineered yeast cells. These cells could perform the desalination task due to their native transport machinery, however, synthetic minimal cells offer a number of significant advantages. Specifically, artificially synthesized cells do not replicate or evolve, hence the mitigated biosafety concerns associated with them. Synthetic cells also display highly predictable behaviors, leading to easier engineering of membrane composition, as well as simplified transport optimization. Lastly, while natural cells devote significant energy to survival and growth, synthetic cells can dedicate nearly all resources to desalination, drastically limiting their metabolic waste.

Overall, the desired outcome of the synthetic cell’s operation would involve the efficient extraction of Na⁺ and Cl^−^ from saltwater, as well as the ions’ stable retention inside the cell. Consequently, the system would produce desalinated water, which could optionally be accompanied by the emission of a fluorescent signal indicating efficient salt removal. All of this would ideally function without consuming too much energy and while ensuring long-term membrane stability, as well as transport activity for extended periods. As an additional feature, the synthetic desalinating cells could be recollected and reused by being physically separated from purified water through filtration or magnetic recovery.

2. Components required for the synthetic cell

As highlighted earlier, membrane-bound compartments are a core requirement for this application. Apart from membrane-anchored transport proteins, building highly stable desalination vesicles requires specialized phospholipids that can stabilize them, effectively mimicking biological ion-transport membranes. Such a membrane should also withstand significant osmotic stress and support ATPase activity, which is essential in moving ions against their gradient. Finally, a reagent that prevents aggregation of the synthetic membranes should be added, as this would maximize the available surface area for ion extraction at all times. Based on these criteria, the lipid or ester components proposed for the desalinating membrane system can be found in Table 9.1.

Table 9.1 Suggested membrane composition of lipids, along with their respective function, for the proposed synthetic cell-based desalination system.

Complementing the lipids that comprise the vesicle’s surface, membrane proteins also play an important role in stabilizing the synthetic cell’s interface with its environment. A large portion of those proteins need to contribute to the cell’s ion-transporting capacity, especially regarding Na⁺ and Cl^−^, or to the cell’s capacity to facilitate the exchange of other equilibrating agents. At this point, before determining which proteins should be included and documenting their coding genes, it is crucial to define the biological context of the system, meaning whether it will be based on a prokaryotic or a more complex eukaryotic cell. For the implementation of the desalination idea, there is a wide variety of membrane proteins transporting ions of both prokaryotic and eukaryotic origin. Since there are no requirements (such as protein glycosylation or post-translational modifications) dictating the more complex internal structure of a eukaryotic cell, a bacteria-based system would suffice in this case and be more energetically efficient, hence the indicative list of membrane proteins originating from prokaryotes presented in Table 9.2.

Table 9.2 List of key transport proteins expressed in the membrane of the synthetic desalinating cell, accompanied by their respective gene and function.

Moving from the surface to the interior of the synthetic cell, all the necessary components of a cell-free Tx/Tl system should be encapsulated within the vesicle’s membrane. As a prokaryotic expression platform has been chosen, using E. coli cell-free Tx/Tl extract seems appropriate. In addition to being widely available and particularly well-characterized, E. coli cell-free Tx/Tl extract is a low-cost, reliable solution, which can provide high membrane protein production and easy scaling. Such a system contains all the necessary machinery necessary for the transcription, translation, and proper folding of the system’s ion-transporting membrane proteins, as described in Table 9.3.

Table 9.3 Main components of a cell-free Tx/Tl system for the expression of membrane proteins, along with their corresponding purpose.

D. Osmoprotectants

High internal salt accumulation creates osmotic stress.

To stabilize the synthetic cell, encapsulate:

These molecules are commonly used by halophilic organisms.

Bulk cell-free Tx/Tl could express transport proteins.

Therefore; combine; logic

• Osmotic regulation
• Artificial membrane engineering

3. ATP-driven pumps concentrate ions internally against their gradients.
4. Internal osmoprotectants stabilize the synthetic cell.

However, membrane proteins are difficult to express.

Therefore:

• Additional chaperones should be included.
• Nanodiscs or detergent-assisted folding may be required.

8. Communication with the Environment

How Will Salt Enter?

Sodium Transport

Na+/K+-ATPase actively imports sodium ions.

Chloride Transport

Halorhodopsin imports chloride ions using light energy.

Water Transport

Aquaporin Z enables rapid water equilibration.

Why Use Halorhodopsin?

Gene: hr Source organism: Halobacterium salinarum

Advantages:

• Light-powered chloride transport
• No ATP required
• Extremely stable in high salt
• Efficient membrane insertion

This reduces external energy requirements.

9. Energy System

ATP Generation

Active ion transport requires ATP.

The synthetic cell would use a hybrid energy system:

Internal ATP Regeneration

• Phosphoenolpyruvate (PEP)
• Pyruvate kinase

Light-Driven Proton Gradient

Bacteriorhodopsin generates a proton gradient. ATP synthase converts this gradient into ATP.

This creates a semi-autonomous energy cycle.

10. Experimental Details

A. Full Lipid List

B. Full Gene List

C. Membrane Protein Reconstitution

Membrane proteins would be inserted using:

• Detergent-mediated reconstitution
• Nanodisc-assisted insertion
• Cell-free co-translational insertion

Nanodiscs improve folding and activity of membrane proteins.

D. Encapsulation Method

Recommended methods:

1. Microfluidic double-emulsion production
2. Water-in-oil emulsion transfer

Microfluidics provides:

• Uniform vesicle size
• High reproducibility
• Controlled protein density

11. Measuring System Function

A. Salinity Measurement

Measure external salt concentration using:

• Conductivity meter
• Ion-selective electrodes
• Flame photometry

Expected result:

• Progressive reduction in external NaCl concentration.

B. Internal Ion Accumulation

Measure internal sodium/chloride using:

• Sodium-sensitive fluorescent dyes
• Chloride-sensitive dyes
• ICP-MS (Inductively Coupled Plasma Mass Spectrometry)

Expected result:

• Increasing ion concentration inside synthetic cells.

C. ATP Measurement

Use luciferase ATP assays.

Purpose:

• Verify active transport energetics.

D. Membrane Integrity

Use:

• Calcein leakage assay
• Rhodamine membrane dyes
• Cryo-electron microscopy

Purpose:

• Confirm vesicle stability under osmotic stress.

E. Protein Expression Validation

Confirm transporter expression using:

• SDS-PAGE
• Western blotting
• Fluorescence labeling

12. Potential Challenges and Proposed Solutions

13. Practical Deployment Strategy

The synthetic cells could be:

1. Immobilized in filtration membranes
2. Packed into microfluidic desalination cartridges
3. Recovered magnetically using embedded nanoparticles
4. Continuously illuminated for energy generation

A practical desalination device would likely combine millions of synthetic cells in parallel.

14. Limitations of the System

Although scientifically plausible, major engineering challenges remain:

• Active transport rates may be too low for industrial desalination.
• ATP generation efficiency is limited.
• Long-term vesicle stability remains difficult.
• Membrane protein integration is technically demanding.
• Synthetic cells may saturate with salt.

Therefore, this system is currently more realistic as:

• A proof-of-concept synthetic biology platform
• A microscale desalination system
• A research tool for artificial cell engineering

rather than a full industrial replacement for reverse osmosis.

15. Broader Applications

The same framework could be adapted for:

• Heavy metal removal
• Radioactive ion capture
• Water purification
• Environmental remediation
• Smart biosensing
• Artificial organelles

By replacing transport proteins, synthetic cells could selectively remove many dissolved contaminants.

16. References

1. Schwille P et al. MaxSynBio: Avenues towards creating cells from the bottom up. Angewandte Chemie International Edition. 2018;57(41):13382-13392.

2. Silverman AD, Karim AS, Jewett MC. Cell-free gene expression: an expanded repertoire of applications. Nature Reviews Genetics. 2020;21:151-170.

3. Rigaud JL, Levy D. Reconstitution of membrane proteins into liposomes. Methods in Enzymology. 2003;372:65-86.

4. Noireaux V, Libchaber A. A vesicle bioreactor as a step toward an artificial cell assembly. Proceedings of the National Academy of Sciences. 2004;101(51):17669-17674.

5. Elani Y, Law RV, Ces O. Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nature Communications. 2014;5:5305.

6. Hwang WL, Chen M, Cronin B, Holden MA, Bayley H. Asymmetric droplet interface bilayers. Journal of the American Chemical Society. 2008;130(18):5878-5879.

7. Gonen T, Walz T. The structure of aquaporins. Quarterly Reviews of Biophysics. 2006;39(4):361-396.

8. Lanyi JK. Bacteriorhodopsin. Annual Review of Physiology. 2004;66:665-688.

9. Kolbe M, Besir H, Essen LO, Oesterhelt D. Structure of the light-driven chloride pump halorhodopsin at 1.8 Å resolution. Science. 2000;288(5470):1390-1396.

10. Lee MT, Sun TL, Hung WC, Huang HW. Process of inducing pores in membranes by melittin. Proceedings of the National Academy of Sciences. 2013;110(35):14243-14248.

11. Kuruma Y, Ueda T. The PURE system for the cell-free synthesis of membrane proteins. Nature Protocols. 2015;10:1328-1344.

12. Murtas G. Artificial assembly of a minimal cell. Molecular BioSystems. 2009;5(11):1292-1297.

13. Phillips R, Kondev J, Theriot J, Garcia H. Physical Biology of the Cell. Garland Science. 2012.

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. How will the idea work, in more detail? Write 3-4 sentences or more. What societal challenge or market need will this address? How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Application Field: Textiles/Fashion One-Sentence Pitch

A smart athletic fabric embedded with freeze-dried cell-free biosensors that activates with sweat to detect dehydration, heat stress, and harmful environmental pollutants in real time through visible color changes.

How the Idea Works

The textile would contain microcapsules filled with freeze-dried cell-free transcription/translation systems integrated directly into the fibers of sportswear or outdoor clothing. When the wearer sweats, moisture rehydrates the cell-free components and activates engineered genetic circuits designed to respond to biomarkers such as sodium concentration, pH, cortisol, or airborne toxins absorbed into sweat. Depending on the detected signal, the fabric would produce visible pigments or fluorescent proteins that change color in specific regions of the clothing. For example, a blue-to-red color shift could warn the user of dehydration, while a fluorescent signal under UV light could indicate dangerous pollution exposure during exercise in urban environments. Because the reactions are cell-free, the system avoids risks associated with living genetically modified organisms while remaining lightweight, flexible, and inexpensive to manufacture.

Societal Challenge or Market Need

This concept addresses the growing demand for wearable health-monitoring technologies that are affordable, noninvasive, and continuously accessible. Athletes, construction workers, military personnel, and people exposed to extreme heat increasingly face dehydration and heat-related illnesses, especially as climate change intensifies global temperatures. Existing wearable electronics often require batteries, sensors, and complex hardware that increase cost and reduce comfort. A biologically integrated textile could provide passive, low-cost physiological monitoring without electronics, making health tracking more accessible in both high-performance sports and low-resource settings. In addition, pollution-sensitive fabrics could help individuals monitor air quality exposure in heavily industrialized or urban areas.

Addressing Limitations of Cell-Free Reactions

Several strategies could improve the practicality of the system despite the limitations of cell-free reactions. To address activation requirements, the system intentionally uses sweat as the hydration trigger, ensuring activation only during wear. Stability could be improved by freeze-drying the Tx/Tl components with protective sugars such as trehalose and embedding them inside hydrogel microcapsules that shield them from oxygen, UV light, and mechanical stress. Because many cell-free systems are single-use, the textile could incorporate replaceable sensing patches or layered fiber compartments that sequentially activate over time, extending garment lifespan. Additionally, low-temperature storage coatings and moisture-resistant packaging could preserve functionality during shipping and storage before use.

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) 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) Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) 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)

Background Information (Maximum 100 words)

Long-duration space missions expose astronauts to increased radiation, microgravity, and confined living conditions, all of which can weaken immune function and promote microbial adaptation. Some bacteria become more stress-resistant and potentially more virulent in microgravity, posing risks to astronaut health and spacecraft environmental safety. Rapid, portable biological monitoring systems are therefore essential for future Moon and Mars missions where conventional laboratory infrastructure is unavailable. This proposal uses the BioBits® cell-free protein expression system to detect activation of bacterial stress-response genes associated with antibiotic resistance and oxidative stress, enabling astronauts to monitor microbial behavior quickly and safely during spaceflight.

Molecular or Genetic Target (Maximum 30 words)

The oxidative stress-response regulator gene oxyR and the antibiotic resistance-associated gene marA in Escherichia coli.

Relationship of Target to the Space Biology Challenge (Maximum 100 words)

Microgravity and space radiation can increase oxidative stress in bacteria, activating stress-response pathways that improve bacterial survival and potentially increase antibiotic resistance. The oxyR gene regulates oxidative stress defense mechanisms, while marA controls multidrug resistance and stress adaptation pathways. Monitoring the expression of these genes provides insight into how bacteria physiologically adapt to spaceflight conditions. Understanding these changes is important because altered bacterial behavior could increase infection risk, reduce antibiotic effectiveness, and compromise astronaut health during long-duration missions where medical resources are limited.

Hypothesis or Research Goal (Maximum 150 words)

This project hypothesizes that simulated spaceflight stress conditions increase activation of the oxyR and marA regulatory pathways in Escherichia coli. The research goal is to develop a rapid, portable, cell-free biosensing workflow capable of detecting bacterial stress-response gene activation during space missions. DNA regulatory sequences responsive to OxyR and MarA activation will be linked to fluorescent reporter expression within the BioBits® cell-free system. If stress-response pathways are activated, the system will produce measurable fluorescence detectable using the P51 Molecular Fluorescence Viewer. This approach would demonstrate that freeze-dried cell-free systems can function as lightweight biological monitoring tools in space without requiring living engineered cells. Such technology could support astronaut health monitoring, spacecraft environmental surveillance, and rapid microbial diagnostics during future deep-space exploration missions.

Experimental Plan (Maximum 100 words)

Escherichia coli cultures exposed to oxidative stress (hydrogen peroxide) will serve as experimental samples, while unstressed cultures will serve as controls. DNA regulatory elements responsive to oxyR and marA activation will drive GFP expression in the BioBits® cell-free system. PCR amplification of target promoter regions will be performed using the miniPCR® thermal cycler before adding templates to freeze-dried reactions. Fluorescence intensity will be measured using the P51 Molecular Fluorescence Viewer. Increased fluorescence in stressed samples compared with controls would indicate activation of bacterial stress-response pathways associated with simulated spaceflight conditions.

!!!!!!!!! There are two major strategies currently used to make cell-free reactions. Some components, like nucleotides and amino acids, can be chemically synthesized. Other components, such as ribosomes and polymerases, still need to be produced by living cells and then separated from the cells. Since scientists have to individually create and purify each component, setting up this type of cell-free reaction is still complex and costly. However, because scientists are able to individually determine every molecule that is put into the reaction, they have tremendous control over the process which can result in high-quality proteins. The second method is to extract all the components directly from host cells all at once. Scientists grow up a large amount of cells and then break them open through a process called lysis. This makes the entire process much simpler and more cost-effective, but it also results in a less purified reaction, as the extract will still contain many unneeded cellular components.

This allows them to learn more and experiment with cellular processes that were previously too difficult to study in living cells. One example is to incorporate non-natural amino acids into the reaction. There are 20 naturally occurring amino acids, but scientists have been able to develop synthetic amino acids with unique chemical properties, and then use these non-natural amino acids to build new proteins in cell-free reactions that cannot be built in natural cells. In 2018, Kazutoyo Miura and their team used nonnatural amino acids to develop a new malaria antigen, which is a small protein that mimics a pathogen used in vaccines to “train” immune systems to fight against specific diseases. The non-natural amino acids in this antigen allow it to bind strongly to immune cells, trigger an immune response, and train them to recognize similar pathogens in the future. With many parts of the world still suffering from malaria and other diseases, we need new vaccines and treatments; using non-natural amino acids may help us discover them. Cell-free reactions don’t have cells that need to be kept alive, but they do contain sensitive molecules that require specific storage conditions. To get around this, scientists freeze-dry the reaction to make them last longer at room temperature. By freezing the reaction and then pulling all of the water out with a vacuum pump, they produce a dry solid that is stable outside of the freezer—similar to how beef left at room temperature will begin to rot, but beef jerky is stable for a long time. All the user has to do is rehydrate their reaction with water, add their DNA of interest, and transcription and translation will begin. Typically, pharmaceutical companies will produce medically-relevant proteins in large batches and ship them on ice to the patients who need them. However, the live-cell production and cold shipping processes are expensive. Freeze-dried, cell-free reactions could be shipped instead so therapeutic proteins can be produced directly in small batches on-demand, virtually anywhere in the world, at a fraction of the cost