Week 9 HW: Cell-Free Systems

General homework questions/answers

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 has several advantages over traditional in vivo protein expression methods. In cell-free systems, researchers have direct control over experimental conditions such as pH, ion concentrations, temperature, substrate availability, and DNA concentration without needing to maintain living cells. This allows for rapid optimization and easier manipulation of gene expression conditions. Cell-free systems also avoid problems associated with cellular toxicity, metabolic burden, and membrane transport limitations. In addition, proteins can be produced much more quickly because there is no need for cell growth or transformation. Cell-free expression is especially beneficial when producing toxic proteins that would kill living cells and when rapidly prototyping genetic circuits for synthetic biology applications.

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

The main components of a cell-free expression system include a cell lysate, DNA template, amino acids, nucleotides, energy source, salts, and cofactors. The cell lysate contains ribosomes, tRNAs, transcription factors, RNA polymerase, and other enzymes required for transcription and translation. The DNA template contains the gene encoding the desired protein product. Amino acids are used as the building blocks for protein synthesis while nucleotides are required for transcription of mRNA. Energy sources such as ATP and phosphoenolpyruvate provide the energy needed for cellular reactions and protein synthesis. Salts and cofactors help stabilize enzymes and maintain proper reaction conditions for efficient protein production.

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 critical in cell-free systems because transcription and translation require large amounts of ATP and GTP. Without continuous energy regeneration, the reaction rapidly loses the ability to synthesize proteins and protein yield decreases significantly. One common method used to maintain ATP levels is the phosphoenolpyruvate (PEP) regeneration system. In this method, phosphoenolpyruvate serves as a high-energy phosphate donor which allows ATP to be continuously regenerated from ADP through enzymatic reactions. Another possible method is the use of creatine phosphate with creatine kinase to recycle ATP 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, such as E. coli lysates, are generally faster, cheaper, and produce high protein yields. However, they lack many post-translational modifications found in eukaryotic cells. Eukaryotic systems, such as wheat germ or rabbit reticulocyte lysates, are slower and more expensive but are capable of producing proteins requiring complex folding and post-translational modifications. A useful protein to produce in a prokaryotic system would be GFP because it folds efficiently and does not require glycosylation or other complex modifications. In contrast, a useful protein to produce in a eukaryotic system would be a monoclonal antibody because antibodies require proper disulfide bond formation and post-translational processing for functionality.

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 membrane protein expression in a cell-free system, I would include lipid nanodiscs, detergents, or artificial liposomes within the reaction mixture to provide a membrane-like environment for proper protein folding and insertion. Membrane proteins are difficult to express because they are hydrophobic and tend to aggregate or misfold outside of membranes. I would also optimize temperature, magnesium concentration, and reaction duration to improve protein stability and translation efficiency. In addition, molecular chaperones could be added to assist with proper folding of the membrane protein during synthesis.

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 degradation of the DNA template or mRNA by nucleases present in the lysate. This issue could be addressed by improving purification methods or adding nuclease inhibitors. A second possible reason is depletion of ATP or other energy substrates during the reaction. This could be solved by improving the energy regeneration system or increasing substrate concentrations. A third possible reason is poor protein folding or aggregation during translation. In this case, lowering the reaction temperature, adding molecular chaperones, or optimizing salt concentrations may improve protein stability and increase overall protein yield.

Homework questions from Kate Adamala

1. Pick a function and describe it.

The function of my synthetic minimal cell would be to detect peanut allergens in food samples and produce a fluorescent glow when peanut allergens are present. This system could be used as a rapid food safety sensor for people with severe peanut allergies.

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

The synthetic cell would sense peanut allergen proteins such as Ara h 1 or Ara h 2 and respond by expressing GFP as a fluorescent output signal. The input would be peanut allergen proteins diffusing into the synthetic cell environment while the output would be green fluorescence visible under blue or UV light.

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

Partially, but encapsulation would significantly improve the system. A purely cell-free system could detect allergens and produce GFP in solution, however encapsulation allows the synthetic cell to better control diffusion, protect internal reaction components, and more closely mimic cellular behavior. Encapsulation also improves stability and enables compartmentalized sensing.

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

Yes. A genetically modified bacterium such as E. coli could potentially be engineered to detect peanut allergens and express GFP. However, synthetic minimal cells are safer because they are nonliving and cannot reproduce or spread in the environment. In addition, synthetic minimal cells avoid many regulatory and biosafety concerns associated with genetically modified organisms.

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

The desired outcome is that the synthetic minimal cells remain inactive in the absence of peanut allergens but rapidly produce visible green fluorescence when peanut allergens are detected. This fluorescence would indicate contamination of the tested food sample with peanut proteins.

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

a. What would be the membrane made of?

The membrane would consist of phospholipids and cholesterol to create a stable lipid bilayer similar to biological membranes. Lipids such as POPC and cholesterol would help maintain membrane fluidity and structural integrity.

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

Inside the synthetic cell I would encapsulate a bacterial cell-free transcription/translation system, ATP regeneration components, amino acids, nucleotides, ribosomes, RNA polymerase, and plasmid DNA encoding GFP under the control of a peanut allergen-responsive regulatory element. I would also include molecular chaperones to improve GFP folding efficiency.

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

A bacterial cell-free expression system derived from E. coli would be sufficient for this application because GFP expression does not require complex mammalian post-translational modifications. In addition, bacterial systems are inexpensive, rapid, and produce high protein yields.

d. How will your synthetic cell communicate with the environment?

The synthetic cell membrane would contain membrane pores such as alpha-hemolysin channels to allow peanut allergen proteins or signaling molecules to diffuse into the synthetic cell. The membrane channels would allow environmental sensing while still maintaining compartmentalization of the internal Tx/Tl machinery.

3. Experimental Details

a. List all lipids and genes.

Lipids: POPC, cholesterol
Cell-free system: E. coli lysate-based Tx/Tl system
Genes: sfGFP reporter gene, alpha-hemolysin (aHL) membrane pore gene, allergen-responsive aptamer targeting Ara h 1/Ara h 2 peanut allergens

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

The function of the system would be measured by detecting GFP fluorescence using a fluorometer or fluorescence microscope. Increased GFP fluorescence would indicate the presence of peanut allergens within the tested sample. Fluorescence intensity could also be quantified over time to measure sensor sensitivity and response speed.

Homework question from Peter Nguyen

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

My concept is a smart architectural paint containing freeze-dried cell-free systems that can detect carbon monoxide gas and produce a visible fluorescent glow to warn building occupants of dangerous conditions.

2. How will the idea work, in more detail?

The paint would contain embedded freeze-dried bacterial cell-free transcription/translation systems along with carbon monoxide-responsive genetic regulatory elements. When carbon monoxide diffuses into the paint layer, the cell-free system would become activated and induce the expression of a fluorescent reporter protein or luminescent enzyme. The glowing signal would provide a rapid visual warning that dangerous carbon monoxide levels are present within the environment. The paint could be applied near furnaces, garages, kitchens, industrial buildings, or enclosed spaces where carbon monoxide leaks are more likely to occur. Because the system is cell-free, there would be no living genetically modified organisms present within the material itself.

3. What societal challenge or market need will this address?

Carbon monoxide poisoning is a major public health issue because the gas is colorless and odorless, making leaks difficult to detect before harmful exposure occurs. This smart paint system could provide an inexpensive and highly visible method for early carbon monoxide detection in homes, schools, factories, and public buildings. In addition, the paint could improve safety in areas where electronic carbon monoxide detectors are unavailable, damaged, or lose power during emergencies.

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

To improve stability, the freeze-dried cell-free components could be encapsulated within protective hydrogel microcapsules embedded throughout the paint. These capsules would protect the biological components from oxygen, UV radiation, and temperature fluctuations while remaining permeable to carbon monoxide gas. Small amounts of environmental humidity could help rehydrate the system when needed, while stabilizing molecules such as trehalose could increase the shelf-life of the biological components. In addition, modular paint layers or replaceable surface coatings could allow the sensing components to be periodically renewed after activation or degradation over time.

Homework question from Ally Huang

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.

As humanity moves toward long-duration spaceflight and possible extraterrestrial colonization, understanding the effects of spaceflight conditions on fetal development becomes increasingly important. Microgravity and ionizing radiation have both been shown to alter gene expression and DNA stability, but their effects on epigenetic regulation during embryonic development remain poorly understood. Improper DNA methylation during development could permanently disrupt growth, organ formation, and viability of embryos in space. Understanding these mechanisms is essential for the future of human reproduction and multigenerational survival during deep-space missions.

2. Name the molecular or genetic target that you propose to study.

DNA methylation patterns and epigenetic regulation of the IGF2 and H19 genes during embryonic development under simulated spaceflight conditions.

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

IGF2 and H19 are imprinted genes that play critical roles in fetal growth and embryonic development. Their expression is tightly regulated through DNA methylation patterns established early during development. Disruption of methylation at these loci can lead to developmental abnormalities and impaired fetal growth. Because microgravity and radiation are known to induce DNA damage and alter cellular regulation, studying methylation changes in IGF2 and H19 could reveal how spaceflight conditions interfere with embryonic development and reproductive success in space environments.

4. Clearly state your hypothesis or research goal and explain the reasoning behind it.

My hypothesis is that simulated spaceflight conditions, specifically microgravity and ionizing radiation, will disrupt normal DNA methylation patterns at the IGF2/H19 imprinting control region, leading to abnormal gene regulation during embryonic development. This hypothesis is based on previous studies demonstrating that radiation exposure and environmental stress can alter epigenetic regulation and DNA methylation stability. Because embryonic development relies heavily on tightly controlled epigenetic programming, even small methylation changes at growth-regulating genes such as IGF2 and H19 may significantly impair fetal development. Understanding these effects would provide insight into the biological limitations of long-term human reproduction in space and help guide future countermeasures for space colonization.

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.

I would expose cultured mammalian embryonic stem cells to simulated microgravity and radiation conditions while maintaining a parallel Earth-gravity control group. DNA samples would be collected and amplified using the miniPCR® thermal cycler. Methylation-sensitive analysis targeting the IGF2/H19 imprinting region would then be performed using BioBits® cell-free systems coupled to fluorescence-based reporters measured with the P51 Molecular Fluorescence Viewer. Fluorescence intensity differences between experimental and control groups would indicate changes in methylation-dependent gene regulation under simulated spaceflight conditions.

Homework Part B: Individual Final Project

I placed my final project slide in the slide deck and submitted the Final Project selection form. I did not end up making a Twist order as I have no lab access. Thus, all my work is done online.