Week 9 HW: Cell-Free Systems

  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.

Main advantages (flexibility & control):

Open system: Components such as DNA, cofactors, salts, inhibitors, can be directly modified. Precise control: You can tune Mg²⁺, ATP, amino acids, etc. Rapid expression: No need for cloning → transformation → growth. Toxic proteins: You can express proteins that would normally kill cells.

When CFPS is better than in vivo:

Producing toxic proteins (e.g., antimicrobial peptides) Studying protein variants quickly (high-throughput screening, mutant libraries) Incorporating non-natural amino acids Expressing membrane proteins without worrying about cell viability

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

Cell extract (lysate): Contains ribosomes, tRNAs, enzymes DNA or mRNA template: The blueprint for your protein Amino acids: Building blocks for protein synthesis Energy system (ATP, GTP + regeneration system): Fuels translation Salts (Mg²⁺, K⁺): Maintain ribosome stability and activity Cofactors (NAD⁺, CoA, etc.): Support metabolic reactions Enzymes (optional): Folding, disulfide bond formation, etc.

  1. 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.

Protein synthesis highly relies on energy. If not enough ATP is available, the system is not able to produce proteins. Without regeneration translation stops quickly, and yield drops dramatically.

For instance, use of phosphoenolpyruvate (PEP) or creatine phosphate is valid as energy sources since these regenerate ATP via substrate-level phosphorylation

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

Prokaryotic systems (e.g., E. coli extract):

Fast, cheap, high yield. However, poor at post-translational modifications

Use case:

Produce enzymes like β-galactosidase → no complex folding/modifications needed

Eukaryotic systems (e.g., wheat germ, insect, mammalian extracts):

Slower, expensive Can do folding, disulfide bonds, glycosylation (depending on system)

Use case:

Produce antibodies or glycoproteins, which need proper folding and modifications

  1. 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.

Challenges include aggregation, misfolding and insolubility. The strategies might include:

Add detergents Use liposomes or nanodiscs to mimic membranes Optimize Mg²⁺ and chaperones Lower temperature to improve folding

A design idea would be:

CFPS + nanodiscs + chaperones → allows co-translational insertion into a membrane-like environment

  1. 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.
  • Poor DNA template quality

Problem: degraded DNA or bad promoter Fix: use fresh plasmid, stronger promoter (e.g., T7), optimize codons

  • Energy depletion

Problem: ATP runs out Fix: improve regeneration system (PEP, glucose system)

  • Protein misfolding or degradation

Problem: aggregates or proteolysis Fix: add chaperones, reduce temperature, include protease inhibitors

  • Suboptimal ion concentrations

Problem: Mg²⁺/K⁺ imbalance kills ribosome activity Fix: optimize salt concentrations experimentally

Homework question from Kate Adamala

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

  1. Pick a function and describe it.

Source: Higashi et. al, 2025

a. What would your synthetic cell do? What is the input and what is the output? It would sense and activate signals in the presence of metals such as mercury for instance. INPUT: Hg²⁺ ions; OUTPUT: AHL (acyl homoserine lactone), a common quorum sensing metabolite.

b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation? No. If not encapsulated, AHL would diffuse freely throughout the medium and not behave like a cell.

c. Could this function be realized by genetically modified natural cell? Yes. However, quorum sensing-related receptors are specific for every metal. Therefore, new cells would have to be modified, which might bring ethical discussions as to whether or not allow such modifications. SMCs, in a way, avoid this.

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

In the presence of SMC: GFP is produced in response to mercury

In the absence of SMC: No response is observed

Thus, the SMC enables bacteria to indirectly sense mercury.

  1. Design all components that would need to be part of your synthetic cell. a. What would be the membrane made of? Phospholipids (POPC) and Cholesterol

b. What would you encapsulate inside? Enzymes, small molecules. It would contain a Tx/Tl system from, for instance. E. Coli, along with the DNA consisting of a promoter with a regulatory protein and a gene (PmerT, MerR and luxI, respectively)

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 promotors, like Tet-ON, you need mammalian) It would be a bacterial system due to it consisting of a metal-responsive system such as MerR and its cost effectiveness.

d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?) The substrate will be permeable since Mercury ions will diffuse through the membrane pores. The ions will activate the genetic circuit that synthesizes AHL, which will help bacteria produce GFP.

  1. 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.)

Lipids POPC Cholesterol

Genes merR PmerT luxI

Cells E. Coli

b. How will you measure the function of your system? The function will be measured through GFP output measurment by flow cytometry.

Homework question from Kate Adamala

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)?

This proposal describes a smart textile integrated with freeze-dried cell-free biosensors capable of detecting airborne pollutants such as carbon monoxide or volatile organic compounds and producing a visible color change as a warning. The system consists of microencapsulated transcription–translation machinery embedded within fabric fibers, which becomes activated upon exposure to environmental humidity (e.g., sweat). When target gases diffuse into the material, they trigger engineered genetic circuits that induce the production of chromoproteins, enabling real-time, user-friendly detection. This technology addresses the growing need for accessible, portable air quality monitoring, especially in urban environments, while limitations of cell-free systems are mitigated through lyophilization with stabilizers, protective encapsulation, and the use of replaceable sensing patches.

Based on Atalie, D., & Fikre, Y. 2026 and Pardee et. al, 2016

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/ .

  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)

Space radiation is a major challenge for long-term human space exploration, as it can damage DNA and compromise biological systems. Current radiation monitoring relies on physical sensors, which do not capture biological effects at the molecular level. Developing biological sensors that directly report DNA damage would provide more relevant information for astronaut health and system performance. Cell-free systems, such as BioBits®, offer a safe, portable, and programmable platform for detecting molecular changes without the need for living cells, making them ideal for space environments where resources and containment are limited.

  1. 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)

DNA damage–responsive regulatory elements (e.g., SOS response promoters such as recA/lexA) controlling expression of a fluorescent reporter (GFP).

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

Space radiation induces DNA damage, particularly double-strand breaks, which activate conserved bacterial DNA damage responses such as the SOS pathway. By incorporating DNA damage–responsive promoters into a cell-free system, it is possible to directly link radiation-induced molecular damage to reporter gene expression. This approach enables real-time biological detection of radiation effects, rather than indirect physical measurements. Such systems are highly relevant for monitoring biomolecular integrity in space habitats, spacecraft, and biological experiments, where understanding functional damage to DNA is critical for maintaining astronaut health and ensuring the reliability of biotechnological systems.

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

The main hypothesis is that DNA damage caused by radiation exposure can be detected in a cell-free system by coupling DNA damage–responsive regulatory elements to the expression of a fluorescent reporter protein. Specifically, if DNA templates or regulatory components are sufficiently affected by radiation, the SOS-like response elements will activate transcription, leading to measurable GFP production. This system would function as a biological dosimeter, translating molecular damage into a quantifiable signal. The reasoning is based on well-characterized bacterial DNA damage responses, which can be reconstituted in vitro using cell-free transcription–translation systems. Demonstrating this capability would support the development of portable, low-resource biosensors for monitoring radiation-induced biological damage during space missions.

  1. 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)

Freeze-dried BioBits® reactions containing DNA constructs with SOS-responsive promoters controlling GFP will be prepared. Samples will include: (1) non-exposed control, (2) radiation-exposed DNA templates, and (3) negative controls lacking promoter activation. DNA may be pre-amplified using miniPCR®. After rehydration, reactions will be incubated and fluorescence measured using the P51 Molecular Fluorescence Viewer. GFP intensity will be compared across conditions to assess activation of the damage-responsive system. Increased fluorescence in exposed samples relative to controls will indicate successful detection of radiation-induced DNA damage.