Week 9 HW: 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 cell production.

Cell-free synthesis excells in enabeling open system manipulation by allowing the direct addition of additives; rapid experimentation and high-throughput since the transformation and cultivation steps can be skiped, moving directly from PCR to protein production; and tolerance to unnatural aminoacids via custom tRNA.

In the case of my final project, cell-free synthesis is more benificial since the first exeriments I would like to perform are based in the synthesis and analisys of chlorophyll-binding light-harvesting proteins, which depend on the presence of chlorophyll to fold properly. If cell-free synthesis is used, chlorophyll can be added as an additive to enable correct folding in a single reaction, otherwise, using cultivation of E.coli, the apoprotein form of the LHP would have to be purified and only after react with chlorophyll.

Another case that I see cell-free synthesis being really usefull is in polymer design, where the possibilities are much more vast and interesting if using de novo amino acids. Cell-free synthesis allows to bipass the in vivo systems that possess established translational machinery adapted for natural occuring amino acids and tRNA.

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

The main components include:

Genetic template either in linear segments or plasmid form
Cell Extract (Lysate) which contains necessary transcriptional/translational machinery: ribosomes, aminoacyl-tRNA synthetases, translation factors, and enzymes. This can be either whole cell extract, which also contain metabolic enzymes and other cellular components typically from E.coli or can be a PURE system which only contains the needed purified machinery.
Buffer which maintains optimal PH for enzymatic activity and reaction stability
ATP that provides energy; GTP, CTP, and UTP contribute to RNA synthesis.
Nucleotides for transcription and Amino acids for translation
Co-folding factors if needed to ensure proper folding of proteins

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 regeneration is essential in cell-free systems because ATP is rapidly consumed during transcription and translation, and without replenishment the reaction quickly stops. One way to maintain ATP levels is to add creatine kinase and phosphocreatine, which regenerate ATP from ADP and provide a continuous energy supply during the experiment.

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

Prokaryotic cell-free systems (e.g., E. coli) are fast, cost-effective, and high-yielding, and allow direct control over reaction conditions, but they lack post-translational modifications. Eukaryotic systems are more complex and lower-yielding, but enable proper folding, disulfide bond formation, and post-translational modifications such as glycosylation.

A prokaryotic system can be used to express, e.g., chlorophyll-binding proteins such as CP43 or PcbA, since chlorophyll can be added directly to the reaction as a folding cofactor, promoting correct assembly of these hydrophobic proteins.

In contrast, a eukaryotic system is suitable for expressing, e.g., fungal lectins (glycoproteins), as their biological activity depends on correct folding, disulfide bond formation, and glycosylation. These modifications are essential for carbohydrate-binding function and are only supported in eukaryotic cell-free systems.

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.

Using chlorophyll-binding membrane proteins such as CP43 or PcbA as example. To optimize their expression in a cell-free system, I would screen different reaction conditions while supplying a membrane-mimicking environment, such as detergent micelles, liposomes, or nanodiscs, so the hydrophobic transmembrane regions can insert properly instead of aggregating. Because these proteins also depend on chlorophyll as a folding cofactor, I would add chlorophyll directly into the reaction to promote correct folding and assembly.

The main challenges are poor solubility, aggregation, misfolding, and low functional yield, since membrane proteins are highly hydrophobic. In the case of CP43 or PcbA, another challenge is that without chlorophyll the protein may not fold correctly or remain stable. To address this, I would optimize variables such as temperature, magnesium concentration, reaction time, membrane mimetics, and chlorophyll concentration, and then evaluate expression by SDS-PAGE/Western blot and functionality by measuring pigment binding or spectroscopic properties. This makes cell-free expression especially useful because the folding environment and cofactors can be controlled directly in one reaction.

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 can arise from degraded or low-concentration DNA, as well as from poorly designed constructs (e.g., weak promoter, inefficient RBS, incorrect spacing, or unfavorable codon usage). These issues reduce transcription and translation efficiency. This can be addressed by using high-quality DNA and optimizing the construct with a stronger promoter, improved RBS, and codon optimization.

Energy depletion or suboptimal reaction conditions, given that cell-free systems rapidly consume ATP and cofactors, which can limit protein synthesis. This can be improved by optimizing the energy regeneration system and adjusting parameters such as Mg concentration, temperature, and reaction time.

Protein misfolding or aggregation, especially for membrane or complex proteins, improper folding can reduce yield. This can be addressed by lowering the reaction temperature, adding chaperones, or including detergents, liposomes, or nanodiscs.

These questions where actually really helpful for my project because while searching for cell-free expression of LHPs and the difficulties of expressing membrane proteins in cell free systems I found WSCPs which are water soluble chlorophyll-binding proteins which provide a promising platform for organizing and stabilizing chlorophyll within aqueous materials, enabling experiments of how protein-mediated pigment structuring affects light-induced degradation processes

Homework question from Kate Adamala

1. Pick a function and describe it: Design a light-responsive synthetic minimal cell that uses chlorophyll to sense light and converts light exposure into a measurable biological signal. This system would function as a biohybrid light sensor or smart material for monitoring sunlight exposure.

What would your synthetic cell do? What is the input and what is the output?: Input: light; Output: production of a detectable reporter signal (e.g., fluorescence or color change) proportional to light exposure Chlorophyll inside the synthetic cell absorbs light and generates a photochemical/redox signal (e.g., reactive oxygen species). This activates a redox-sensitive transcription factor, e.g. OxyR, which triggers Tx/Tl of a reporter protein such as GFP or an enzyme that produces a colored product.
Could this function be realized by cell-free Tx/Tl alone, without encapsulation?: Only partially. In a bulk reaction, the signal would diffuse and lack spatial organization. Encapsulation is important because it creates discrete microreactors, protects the components, and allows integration into materials such as coatings or hydrogels.
Could this function be realized by genetically modified natural cell?: Yes, but synthetic cells are preferable because they are non-living, easier to control, and more compatible with material applications. Natural cells introduce metabolism, growth, and variability that can interfere with stable sensing.
Describe the desired outcome of your synthetic cell operation: Upon illumination, synthetic cells produce a signal proportional to light exposure. When embedded in a material, they form a light-responsive layer that can map or record light intensity.

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

What would the membrane be made of?: Phospholipids + cholesterol
What would you encapsulate inside?: E. coli cell-free Tx/Tl system; DNA encoding: OxyR (redox-sensitive transcription factor); reporter protein (e.g., GFP or luciferase); chlorophyll; amino acids, NTPs, ATP regeneration system
Which organism your Tx/Tl system will come from?: A bacterial (E. coli) cell-free system is sufficient, as no complex post-translational modifications are needed.
How will your synthetic cell communicate with the environment?: Light freely crosses the membrane and activates chlorophyll. Small molecules such as oxygen can diffuse through the membrane. If enhanced exchange is required, a membrane pore such as α-hemolysin (aHL) can be included.

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

List all lipids and genes: Lipids— POPC; cholesterol. Genes— oxyR (E. coli); gfp or luciferase gene
How will you measure the function of your system?: Measure fluorescence (GFP) or luminescence (luciferase); Compare signal across different light intensities; Quantify output as a relation of exposure time

Homework question from Peter Nguyen

Develop a system that allows the transformation of almost any surface into a photographic support through the microencapsulation of a freeze-dried cell-free system embedded within a polymer matrix. This system would remain inert in the dry state and be activated upon exposure to moisture, initiating a series of biochemical and photochemical processes that generate light-sensitive chlorophyll domains. Rather than synthesizing chlorophyll de novo, the system would incorporate pre-encapsulated chlorophyll or chlorophyll–protein complexes, stabilized within the material. Upon hydration, the cell-free system would become active and could produce supporting components (such as stabilizing proteins or enzymes) that help organize or maintain these pigment domains.

Once activated, the surface would gradually develop a more visible green coloration, indicating the presence of functional chlorophyll domains. When exposed to light, chlorophyll would undergo photodegradation into derivatives capable of chelating iron ions present in the surrounding matrix. These reactions would lead to the formation of a permanent image, effectively allowing the surface to “self-develop” without the need for external chemical processing.

The system could be tuned to respond to specific wavelengths of light, enabling controlled image formation while minimizing unwanted degradation. Visually, one could imagine walls or textiles that, once hydrated, slowly become photosensitive, capture light patterns over time, and then darken as the iron-based reaction fixes the image.

The idea behind this system— chlorophyll-based analog photography— has the main objective to substitute the toxic silver-based chemistry in current photographic emulsions. This application could be a way of demonstrating this technology, since photographic technology is usually a Blackbox. Chlorophyll Photographic Surfaces could be an amazingly visual way to observe the fascinating chemistry of chlorophyll as it captures a moment in time.

The limitations of cell-free systems are treated as design features. Activation by water allows the material to remain stable in its dry state and only become functional upon hydration, preventing premature chlorophyll degradation. Stability is ensured through freeze-drying and microencapsulation within a polymer matrix, which protects the components during storage. The one-time-use nature of the system aligns with its role as a photographic process, where a single, irreversible transformation is required to record and fix an image.

Homework question from Ally Huang

Background: Spaceflight exposes astronauts to increased radiation, which leads to the production of reactive oxygen species (ROS) and oxidative stress. This can damage DNA, proteins, and cellular function, posing risks to astronaut health during long-duration missions. Understanding how oxidative stress affects biological systems is therefore critical for developing protective strategies. Cell-free systems provide a simplified platform to study these effects without the complexity of living cells. This project proposes using a cell-free protein expression system to model how oxidative stress influences gene expression, providing insight into biological damage mechanisms relevant to space environments.

Molecular / genetic target: OxyR transcription factor and an OxyR-responsive promoter controlling GFP expression.

Relation to space biology: Radiation in space generates reactive oxygen species (ROS), which induce oxidative stress in biological systems. The OxyR transcription factor in bacteria is activated by oxidative conditions and regulates gene expression in response to ROS. By coupling OxyR activation to GFP expression in a cell-free system, this experiment models how space-induced oxidative stress affects transcriptional responses. This provides a simplified and controllable way to study how oxidative conditions influence gene regulation and molecular damage in space.

Hypothesis / research goal: The hypothesis is that exposure to space-induced oxidative stress will activate OxyR, resulting in increased GFP expression up to a threshold, beyond which excessive oxidative damage will reduce protein production. This is based on the known mechanism of OxyR, which is activated by oxidation, enabling it to promote transcription of target genes. However, high levels of oxidative stress can damage transcriptional and translational machinery, reducing overall protein synthesis. The goal of this experiment is to characterize how oxidative stress in space affects gene expression in a cell-free system and to identify conditions where biological systems remain functional versus when damage becomes inhibitory.

Experimental plan: BioBits cell-free reactions containing OxyR and GFP under an OxyR-responsive promoter will be prepared and flown to the ISS. Samples will be exposed to the space environment, where radiation is expected to generate oxidative stress. Parallel ground controls will be maintained on Earth. Additional onboard controls will include reactions lacking OxyR or containing constitutive GFP expression. Fluorescence will be measured using the P51 Molecular Fluorescence Viewer to quantify GFP production. The miniPCR may be used to amplify DNA templates if needed. Differences between flight and ground samples will reveal how space-induced oxidative stress affects gene expression.

References

Molecular Cloning and Functional Expression of a Water-soluble Chlorophyll Protein

The pigment binding behaviour of water-soluble chlorophyll protein (WSCP)

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose

Structural details of the OxyR peroxide-sensing mechanism

Radiation risk mitigation in human space exploration