Week 9 HW: Cell-Free Systems

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General Homework Questions

Question 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.

Answer

Cell-free protein synthesis is a biotechnology technique for producing proteins in a test tube using biological machinery extracted from a cell.

In terms of flexibility, cell-free protein synthesis is an open reaction environment. This allows for the direct addition, removal, and modification of any reaction component at any time. Additionally, unlike in living cells, there are no constraints related to cell viability, enabling the rapid prototyping of genetic constructs, testing different pH and temperature conditions, and the expression of proteins that would be toxic or lethal to living cells. In terms of control over experimental variables, in cell-free protein synthesis. Each component of the reaction, such as ATP concentration, pH, and amino acid ratios, can be independently tuned with exact precision, enabling more reproducible and targeted biological investigations.

Cell-free expression is more beneficial when producing proteins that would be lethal to host cells, incorporating non-natural amino acids, and for on-demand protein production, such as in portable biosensors. Additionally, it speeds up the testing of genetic constructs and designs by eliminating the waiting period for cloning and cell growth.

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

Answer

The main components of a cell-free expression system are:

  • Cell extract (Lysate): It is the core of the system and provides the biological machinery needed to convert genetic information into protein. It is derived from organisms and contains ribosomes to carry out protein synthesis, tRNAs to deliver amino acids to the ribosomes, and enzymes to support transcription, translation, and energy metabolism.

  • DNA or mRNA templates: They encode the protein of interest and serve as the blueprint for protein synthesis.

  • Energy Source and regeneration system: It supplies the energy required for transcription and translation, as well as regenerates Adenosine Triphosphate (ATP) and Guanosine Triphosphate (GTP) from Adenosine Diphosphate (ADP) and Guanosine Diphosphate (GDP). To maintain high-efficiency protein production for hours. It includes molecules like ATP, GTP, and energy substrates such as Phosphoenolpyruvate and creatine phosphate.

  • Amino acids: They are the building blocks used to assemble proteins by the ribosome.

  • Cofactors and Salts: They maintain optimal conditions for enzyme activity, ribosome stability, and overall reaction efficiency. They usally inculde magnesium ions (Mg2) and potassium ions (K+).

Question 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.

Answer

Energy provision regeneration is important in cell-free systems because transcription and translation, which drive protein synthesis, are energy-intensive processes that quickly deplete ATP present in the system. Therefore, efficient energy provision regeneration is required to prevent premature reaction termination and low protein yields.

An effective method to ensure continuous ATP supply is to use a phosphoenolpyruvate (PEP) based energy regeneration system. In the method, PEP acts as a high-energy phosphate donor in the presence of pyruvate kinase and transfers a phosphate group to ADP to regenerate ATP, providing the system with energy.

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

Answer

Prokaryotic cell-free expression systems offer low cost, high and faster yield of proteins that are easy to optimize. However, they do not possess the cellular machinery for complex post-translational modifications such as advanced protein folding and glycosylation. On the other hand, eukaryotic cell-free expression systems closely mimic the environment of higher organisms and support the folding of complex proteins and membrane protein insertion, but are more expensive and slower compared to prokaryotic systems.

For a prokaryotic cell-free system, I would choose the green fluorescent protein (GFP) because it is a simple protein and does not require complex post-translational modifications such as glycosylation to function.

For a eukaryotic cell-free expression system, I would choose the Human Erythropoietin (EPO), which is a glycoprotein responsible for the production of red blood cells in the bone marrow. It requires glycosylation to function properly and is ideal for a eukaryotic cell-free expression system.

Question 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.

Answer

A membrane protein is a protein molecule that is attached to or interacts with the membrane of a cell or organelle. It usually acts as a transporter, receptor, or enzyme that enables cells to communicate, obtain nutrients, and manage energy.

To optimize the expression of a membrane protein in a cell-free experiment, I would first consider an appropriate system to obtain the cell-free lysate. I would use a cell lysate from E. coli; it is cheap, has high yield, and would enable rapid screening by serving as a high-throughput testing platform. I would later use a eukaryotic cell lysate if the membrane protein requires post-translational modifications for its functionality. I would then design a DNA construct with a strong promoter for the detection of the protein.

I would add detergents, liposomes, or nanodiscs to the reaction to provide a membrane-like environment to support proper protein folding and co-translational insertion. The cell-free reaction would also include molecular chaperones and energy regeneration systems to enhance folding and sustain protein synthesis. I would then perform parallel reactions with variables such as lipid composition, detergent type, DNA concentration, and temperature varied. I would evaluate the expression levels, solubility, and functionality of the membrane protein, selecting the best-performing conditions and refining them to maximize yield while ensuring proper folding and function of the membrane protein.

A challenge I would face during the experiment is protein aggregation due to hydrophobic transmembrane regions clamping together in aqueous environments. I would overcome this by including detergents, liposomes, or nanodiscs to provide a stable lipid environment. Another challenge is improper folding due to a lack of a full cellular machinery for correct protein conformation. I would address this challenge by adding a molecular chaperone and lowering the reaction temperature to promote proper protein folding. A final challenge would be low yield, which is common among membrane proteins due to their instability and translational difficulty. I would address the issue of low yield by adjusting ionic conditions, DNA concentrations, and maintaining efficient energy systems to ensure continuous protein synthesis.

Question 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.

Answer

In a cell-free system the low yield of a target protein may be due to the following reasons:

  • Inefficient transcription or translation from the DNA template, this may be due to issues with the DNA construct, such as a weak promoter or poor ribosome binding site. Additionally, suboptimal codon usage or degraded DNA templates could also significantly impact transcription and translation.

Troubleshooting strategy: Redesign the DNA construct with a strong promoter and an optimized ribosome binding site. Additionally, mRNA can be used directly instead of DNA to bypass the limitations of transcription.

  • Energy Depletion due to an insufficient energy regeneration system or insufficient ATP or GTP, which halts the reaction.

Troubleshooting strategy: The energy regeneration system should be optimized fr the reaction or supplemented with additional ATP, GTP, and cofactors.

  • Protein misfolding or aggregation may occur if the protein is too large, complex, or membrane-associated, which leads to a low functional yield.

Troubleshooting strategy: Lowering the temperature of the reaction can improve folding, and the inclusion of molecular chaperones, detergents, liposomes, or nanopores can also aid in improving folding in membrane proteins.

Homework Question from Kate Adamala

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

Question 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 a genetically modified natural cell? d. Describe the desired outcome of your synthetic cell operation.

Question 2. Design all components that would need to be part of your synthetic cell. a. What would the membrane be made of? b. What would you encapsulate inside? Enzymes, small molecules. c. 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 promoters, like Tet-ON, you need mammalian) d. How will your synthetic cell communicate with the environment? (Hint: are substrates permeable? or do you need to express the membrane channel?)

Question 3. Experimental details

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

Answer

Question 1.

a.

I would design a synthetic microbial cell that detects heavy metal contamination(lead, arsenic, mercury, and cadmium) in water samples from galamsey-affected communities. The input is river water possibly containing dissolved heavy metal ions at varying concentrations, and the output will be distinct colorimetric signals for each pollutant: blue for lead, orange for arsenic, purple for mercury, and brown for cadmium. The color will be produced inside the vesicle and visible through the membrane, making it readable by the naked eye.

b.

Yes, it can be realized without encapsulation in a cell-free Tx/TL system by directly exposing the reaction mixture to the water sample to be tested. This will allow the metal ions to interact with the sensing proteins to trigger reporter expression.

c.

Yes, its function can be realised by a genetically modified natural cell, such as a modified E. coli strain. The modified E. coli strain would express PbrR, ArsR, MerR, and CadC coupled to a colorimetric reporter.

d.

The desired outcome is that the synthetic cell produces a visible, distinct colorimetric signal in the presence of lead, arsenic, mercury, and cadmium, indicating which metals are present in the river sample.

Question 2.

a.

The synthetic cell will have a membrane composed of a phospholipid bilayer that is permeable to small ions but can retain DNA, ribosomes, and proteins inside. It could have a possible lipid composition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (POPG), and Cholesterol. POPC would provide bilayer stability and fluidity across a wide temperature range, while POPE would add mechanical stability and reduce the membrane’s permeability to unwanted solutes. POPG will add a negative surface charge to stabilize the vesicle and improve its compatibility with the encapsulated E. coli Tx/Tl machinery. Cholesterol will modulate the fluidity of the membrane and reduce passive permeability to ions to ensure the entry of metal ions is controlled through expressed membrane channels rather than passive diffusion.

b.

The synthetic cell would encapsulate:

  • Genetic material for two plasmids that contain PbrR-LacZ + ArsR-CrtI circuits and MerR-BpsA + CadC-MelA circuits for heavy metal detection.
  • Tx/Tl machinery from E. coli.
  • A phosphocreatine and creatine kinase energy regeneration system to sustain the synthesis of the reporter proteins.
  • Heavy metal-sensing proteins: PbrR protein (lead sensor), ArsR protein (arsenic sensor), MerR protein (mercury sensor), CadC protein (Cadmium sensor).
  • Reporter substrates for each reporter protein.
  • Buffer and salts.

c.

The Tx/ Tl system will come from the E. coli BL21 (DE3) bacterial system. This is because the four metal-sensing proteins are of bacterial origin and function optimally in a bacterial biochemical environment. Additionally, the T7 promoter driving the circuits is compatible with the E. coli Tx/Tl machinery.

d.

The synthetic cell will communicate with the environment by incorporating specific membrane ion transporters for each target metal. To overcome the impermeability of the lipid bilayer to charged metal ions, which would prevent them from reaching the encapsulated sensing proteins. The possible transporters are the lead transporter protein (PbrT), the Glycerol uptake facilitator protein (GlpF) for arsenite entry, the mercuric transporter protein (MerT), and the CadA-associated transporter.

Question 3.

a.

Lipids:

  • POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)
  • POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)
  • POPG ( 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol),)
  • Cholesterol

Genes:

  • pbrR (Lead sensor)
  • arsR (Arsenic sensor)
  • merR (Mercury sensor)
  • CadC (Cadmium sensor)
  • LacZ (β-galactosidase) Blue color
  • crtl (phytoene desaturase) Orange color
  • bpsA (Indigodine synthetase) purple color
  • melA (Laccase/ melanin) Brown color
  • pbrT (Lead membrane transporter)
  • glpF (Arsenite channel)
  • merT (Mercury membrane transporter)
  • mntH (Divalent cation importer (Cd²⁺))

b.

I will measure the function of the system through the appearance of a distinct color inside or outside the vesicles of the synthetic cells in correspondence to the detection of a heavy metal pollutant. Additionally, I can use an inductively coupled plasma mass spectrometry (ICP-MS) to measure both internal and external metal ion concentration after incubation to confirm the uptake and accumulation of metal ions.

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.

Answer

A band-aid or wound dressing with embedded freeze-dried cell-free circuits that would sample wound exudate, to detect the molecular signatures of specific bacterial pathogens, and in response produce and release precise antimicrobial peptides needed to counter the pathogens and prevent the infection of the wound.

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

Answer

The band-aid or dressing would have three layers. The innermost layer will be in contact with the wound and wick fluid outward through capillary action, in a similar way to how flow test strips work. The middle layer will be the core and contain freeze-dried cell-free microzones that are each programmed to recognize a different bacterial pathogen by detecting its unique RNA signature. When wound fluid rehydrates a zone and a matching pathogen RNA is present, the circuit switches on and produces both the specific antimicrobial peptide that kills that exact bacteria, and a visible color unique simultaneously. The outermost layer will be a breathable waterproof backing that retains moisture to keep the chemistry running. The colour will be displayed on the surface of the outermost layer for clinicians to read.

What societal challenge or market need will this address?

Answer

This idea would address the rise of antimicrobial resistance by enabling immediate pathogen-specific treatment through the dressing/ band-aid, which will eliminate the need for broad-spectrum antibiotics and delays associated with traditional diagnostic procedures. Additionally, it would also improve the outcomes for chronic wound patients by enabling continuous monitoring and targeted therapy to prevent infections and lower healthcare costs.

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

Answer

For this idea, activation with water would not be a limitation but rather a function of the design since wound fluid is the activation trigger of the band-aid/ dressing. The ability of the band-aid/ dressing would be ensured by freeze-drying and protective encapsulations that would preserve its sensitive components until use. The one-time use of cell-free systems would not be a limitation here but align with standard wound care practices, with each dressing delivering a fresh, effective dose.

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/ .

Question 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)

Answer

Astronauts are exposed to ionizing radiation such as cosmic rays and solar particle events during space exploration. Their exposure to these doses of radiation, which are almost 100 times higher than what reaches the Earth’s surface, does not imbue them with superpowers but causes cumulative DNA double-stranded breaks. This increases cancer risk and threatens the mission integrity of long-term space exploration missions. Currently, during space missions, radiation damage is monitored through passive dosimetry bags that measure only physical damage. I would like to propose the development of a rapid, low-resource cell-free tool to measure biological damage due to radiation exposure in real time.

Question 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)

Answer

The target molecule would be phosphorylated histone H2AX (γH2AX). It is an early molecular marker for DNA double-stranded breaks and replication stress. It is detectable in blood, urine, and saliva. I think collecting saliva samples will be the best way to measure γH2AX.

Question 3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

Answer

When ionizing radiation causes a double-stranded break in DNA, the cells trigger an emergency alarm and rapidly tag the nearby H2AX protein by attaching a phosphate group to it and convert it into γH2AX. γH2AX acts like a flare planted directly at the damage site with more DNA breaks, meaning more flares. Therefore, measuring can tell us exactly how much radiation damage an astronaut’s cells have suffered.

Question 4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

Answer

My hypothesis involves using Biobits as a cell-free expression system to encode for an anti-γH2AX nanobody fused to a split fluorescent reporter to detect relevant concentrations of γH2AX in astronauts’ saliva samples to provide a quantitative, real-time index of cumulative DNA damage during spaceflights.

The reasoning behind this is that current diometry tells mission controllers how much radiation has been absorbed by the spacecraft. However, it cannot tell how much DNA damage a particular astronaut has sustained in a week versus last week. As identical doses of radiation may produce differing biological outcomes in individual astronauts due to their varying DNA repair capacity. A personalized repeated biological readout that would allow for dynamic mission decisions, such as flagging crew members with unexpectedly high DNA damage accumulation for reduced exposure duties, and aid in building a longitudinal biological dataset to set evidence-based radiation exposure limits for possible future deep space exploration missions.

Question 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)

Answer

I would collect saliva from the astronauts before, during, and after high radiation events, and saliva collected from before going into space will serve as the control. The saliva will be added to rehydrated Biobits reactions encoding an anti-γH2AX nanobody split green fluorescent protein fusion. The γH2AX in the samples will bridge split GFP halves, reconstituting fluorescence proportional to γH2AX concentration. The intensity of the fluorescence measured will be used to plot a γH2AX concentration curve over the mission duration for each crew member.

Homework Part B: Individual Final Project

Question 1. I have added my final project slide to the committed listener ONE FINAL PROJECT IDEA deck.

Question 2. I have submitted the final project selection form.

Question 3. I have also begun planning how to write my final project documentation based on the HTGAA project guidelines.

Question 4. I have begun preparing my first DNA order.

Reference

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Membrane Proteins. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26878/

Calhoun, K. A., & Swartz, J. R. (2007). Energy systems for ATP regeneration in cell-free protein synthesis reactions. Methods in molecular biology (Clifton, N.J.), 375, 3–17. https://doi.org/10.1007/978-1-59745-388-2_1

Gregorio, N.E., Levine, M.Z. and Oza, J.P., 2019. A user’s guide to cell-free protein synthesis. Methods and protocols, 2(1), p.24.

Mah, LJ., El-Osta, A. & Karagiannis, T. γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24, 679–686 (2010). https://doi.org/10.1038/leu.2010.6

Mason, E. (2023) Advantages of cell-free protein expression. Biocompare. Available at: https://www.biocompare.com/Editorial-Articles/594727-Advantages-of-Cell-Free-Protein-Expression/?catid=7123 (Accessed: 5 April 2026).

Osaki, T. and Takeuchi, S., 2017. Artificial cell membrane systems for biosensing applications. Analytical chemistry, 89(1), pp.216-231.

Sachse, R., Dondapati, S.K., Fenz, S.F., Schmidt, T. and Kubick, S., 2014. Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes. FEBS letters, 588(17), pp.2774-2781.

Sharma, B., Moghimianavval, H., Hwang, S. W., & Liu, A. P. (2021). Synthetic Cell as a Platform for Understanding Membrane-Membrane Interactions. Membranes, 11(12), 912. https://doi.org/10.3390/membranes11120912

Yue, K., Zhu, Y., & Kai, L. (2019). Cell-Free Protein Synthesis: Chassis toward the Minimal Cell. Cells, 8(4), 315. https://doi.org/10.3390/cells8040315