Week 9 HW: Cell-Free Systems

General Homework Questions

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 systems are advantageous for quick, high-throughput protein production. Cell-free protein expression is more time efficient than traditional in vivo methods because they do not require the cloning steps of cell-based systems. Additionally, cell-free systems can be modified to include non-canonical amino acids which enables numerous biotechnology and pharmaceutical applications. Cell-free systems also offer the advantage of ease of manipulating reaction conditions. Cell-free expression is more beneficial than cell production when evaluating proteins that are toxic to a cell or when a large yield of the desired protein is needed in a relatively short period of time.

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

• Genetic template encoding the desired protein (DNA or mRNA)
• Energy source (ATP)
• RNA polymerase and nucleotides (if DNA template, for mRNA synthesis)
• tRNA, amino acids, and ribosomes (for translation)
• Enzymatic cofactors

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 is critical in cell-free systems because the energy source (ATP) powers both transcription and translation. One method that could be used to ensure a continuous supply of ATP in a cell-free experiment is the “protein synthesis using recombinant elements” (“PURE”) system which generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP.

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

Prokaryotic cell-free expression systems are ideal for simple, high-yield protein synthesis with limited post-translational modifications. Eukaryotic systems are lower yield and higher cost, but they support the production of more complex proteins that undergo processing through the eukaryotic endomembrane system. An ideal protein for prokaryotic cell-free protein expression might be an enzyme such as beta-galactosidase which supports E coli metabolism by breaking lactose into glucose and galactose monomers. An example of a protein that may be produced in a eukaryotic cell-free system would be an antibody because antibodies require proper folding and assembly to be functional.

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.

I would work with a eukaryotic cell-free expression system because membrane proteins require complex folding which is more suited for a eukaryotic system. One challenge in this experiment might be aggregation of hydrophobic proteins (the exterior of a protein’s transmembrane domain is often hydrophobic) which may inhibit proper folding.

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.

1. The promoter and RBS are not well-suited to the expression system - change to a promoter of different strength
2. Insufficient energy regeneration - adjust energy regeneration system to ensure adequate ATP is produced to carry out cell-free protein production
3. Protein misfolding or aggregation - modify reaction conditions or incorporate molecular chaperone (or other proteins that facilitate proper folding) into the system

Homework question from Kate Adamala

Pick a function and describe it.

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

My synthetic cell would detect a cancer-associated molecular signal and respond by producing a cytotoxic protein. The input would be a protein associated with tumorigenesis and the output would be a cytotoxic protein, such as DTA.

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

A cell-free system could be used to express DTA, but without a cell membrane the reaction would not be spatially contained and the DTA toxin could diffuse everywhere (to non-tumor cells).

Could this function be realized by genetically modified natural cell?

Yes, engineered cells (ex. CAR-T cells) can detect tumor markers and express cytotoxic proteins.

Describe the desired outcome of your synthetic cell operation.

The circuit in the synthetic cell would not be activated until a tumor-associated molecular signature is detected. Once activated, the cell would produce and release DTA to target neighboring tumor cells.

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

What would be the membrane made of?

The membrane would be comprised of lipids that compose a standard cell (phospholipids, cholesterol, etc).

What would you encapsulate inside? Enzymes, small molecules.

The plasmid containing the input-responsive sensor and DTA gene, cell-free system components (see problem above for complete list), and the necessary buffers and co-factors for enzymatic function.

Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason?

Some mammalian lysate that is genetically siilar enough to mimic the environment and support the folding of human proteins.

How will your synthetic cell communicate with the environment?

The circuit within the synthetic cell will be sensitive to tumor-associated proteins within the cellular environment and will release the cytotoxic DTA in a controlled manner into the cellular environment.

Experimental details.

List all lipids and genes.

Lipids: phospholipids, cholesterol Genes: sensor gene (sensitive to tumor-associated protein) and DTA gene

How will you measure the function of your system?

I could incorporate a reporter fluorescence gene or measure the concentration of DTA in the cellular environment.

Homework question from Peter Nguyen

Write a one-sentence summary pitch sentence describing your concept.

A freeze-dried system that can coat the barriers of aquatic environments (i.e. aquarium displays, fish tanks) to visually signal when environmental conditions become hazardous (i.e. pH becomes too acidic or basic to support plant and animal life, algal bloom, etc.).

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

The coating consists of a thin hydrogel layer embedded with micro‑domains of freeze‑dried cell‑free reactions. Each micro‑domain contains a pH‑responsive genetic circuit that activates expression of a visible chromoprotein when the surrounding water becomes too acidic or too basic. When the aquarium water contacts the surface, the cell‑free spots rehydrate and begin sensing; if the pH crosses a preset threshold, the chromoprotein is produced and the coating changes color. Different regions can be tuned to different pH ranges, creating a spatial “map” of water quality across the structure.

What societal challenge or market need will this address?

Aquariums, aquaculture systems, and aquatic research facilities rely on stable water chemistry to keep organisms healthy, but existing monitoring tools can be expensive, technical, or require constant calibration. A passive, visually intuitive biosensing surface would make water‑quality monitoring more accessible for hobbyists, educators, and public installations. This reduces the risk of unnoticed pH drift, which is a major cause of stress and mortality in aquatic organisms.

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

This system is intentionally water-activated (designed for aquatic environments). To promote stability, protective components will be embedded in the hydrogel matrix to protect against light damage or oxidation. To protect against one-time use, the hydogel film could be multi-layered or coated on a replacable panel.

Homework question from Ally Huang

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.

Long‑duration spaceflight increases the risk of nutrient deficiencies and immune changes because astronauts have limited access to fresh food and experience altered metabolism in microgravity. Vitamin D pathways are especially affected due to minimal UV exposure and changes in bone physiology. Transporting large quantities of supplements is mass‑limited, so an on‑demand system for producing beneficial proteins would improve crew health and mission resilience.

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.

A DNA construct encoding vitamin D–binding protein (DBP) fused to a fluorescent reporter, enabling simultaneous production and visualization of a nutritionally relevant protein.

Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses.

Vitamin D metabolism is disrupted in space due to reduced UV exposure and altered calcium homeostasis, contributing to bone loss and immune changes. Producing DBP in situ serves as a model for generating nutritional or therapeutic proteins from dry‑stored DNA during long missions. The fluorescent fusion allows astronauts to directly monitor expression using the P51 fluorescence viewer, making it easy to compare performance in microgravity versus Earth. This target links a concrete health concern to a generalizable strategy for on‑demand biomanufacturing in space.

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

Freeze‑dried cell‑free reactions can reliably express a functional DBP–fluorescent fusion protein under spaceflight conditions, demonstrating the feasibility of on‑demand production of beneficial proteins during long‑duration missions. Microgravity and space radiation may influence reaction kinetics, folding efficiency, or stability, but that properly formulated cell‑free reactions will retain sufficient activity for practical use. The research goal is to quantitatively compare expression yield and fluorescence intensity of the DBP fusion protein in space versus matched ground controls. Establishing robust expression in microgravity would support future development of compact “protein pharmacy” kits for astronauts, enabling flexible nutritional supplementation or rapid production of medically relevant proteins without relying solely on pre‑packaged supplies.

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.

Freeze‑dried cell‑free reactions containing the DBP–fluorescent fusion plasmid will be prepared alongside negative (no DNA) and positive (standard fluorescent protein) controls. Astronauts will rehydrate reactions with buffer, incubate them at ambient ISS temperature, and measure fluorescence using the P51 fluorescence viewer. Identical ground controls will be run with the same lots and timing. Data collected will include fluorescence intensity over time and qualitative observations of reaction color. Comparing space and Earth results will reveal how microgravity and storage affect cell‑free protein expression.