Week 9 HW: Cell Free Systems

Homework Part A: General and Lecturer-Specific Questions

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.

The primary advantage lies in the decoupling of the reaction from cellular metabolism. ​Flexibility: It allows the use of linear DNA, eliminates the need for transformation and host-specific codon optimization, and facilitates the expression of proteins that are toxic to the host. ​Control of variables: It is an “open” system. You can manipulate buffer composition (pH, ionic strength), add chaperones, modify the Mg2+/K+ ratio, or add specific redox agents for disulfide bond formation in real-time, without the limitations of cellular homeostasis. ​Use cases: ​Toxic proteins: Production of proteins that compromise host viability (e.g., antimicrobial peptides or nucleases). ​Non-canonical amino acid (ncAA) incorporation: Facilitates genetic code expansion via stop codon suppression without competition from endogenous tRNAs.

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

​Cell lysate: Source of translational machinery (ribosomes, tRNAs, initiation/elongation factors, aminoacyl-tRNA synthetases). ​Reaction buffer: Salts (K+, Mg{2+}), nucleotides (NTPs), and amino acids. ​Energy regeneration system: (See point 3). ​DNA template: Plasmid or linear DNA with strong promoters (e.g., T7). ​RNA polymerase: Typically T7 RNA polymerase if the template is specific

  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.

Energy provision regeneration is critical because ATP is rapidly consumed by protein synthesis and amino acid activation; furthermore, the accumulation of inorganic phosphate (Pi) inhibits the system. ​Suggested method: Phosphoenolpyruvate (PEP)/Pyruvate kinase system. PEP acts as a high-energy phosphate donor to regenerate ATP from ADP, maintaining a stable ATP/ADP ratio. Alternatively, Creatine phosphate/Creatine kinase is used for a slower, less toxic release kinetic.

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

​Prokaryotic (E. coli): High yield, simple, cost-effective. Ideal for cytosolic proteins. ​Eukaryotic (Wheat germ/Rabbit reticulocyte/HeLa): Allows for complex folding, glycosylation, and protein complex formation requiring mammalian-specific chaperones. ​Selection: For a human membrane protein, I would choose HeLa or rabbit reticulocyte lysate, as they provide the lipid environment (micelles or vesicles) and chaperones necessary for correct membrane folding, which E. coli cannot efficiently replicate. 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. ​Membrane proteins are hydrophobic and prone to aggregation/precipitation outside a lipid environment. ​Design: Add nanodiscs (MSP - Membrane Scaffold Proteins) or detergents (Brij-35, Triton X-100) to the lysate. These provide a hydrophobic surface where the protein can insert co-translationally. Strategy: Optimize Mg2+ concentration (crucial for correct insertion) and perform the reaction at reduced temperatures (25–30°C) to slow down translation and allow for proper folding.

  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.
  • Template degradation: DNA is attacked by endogenous nucleases in the lysate. Strategy: Use recBCD- strains deficient in exonucleases or add nuclease inhibitors.
  • Byproduct accumulation: Pi inhibits the reaction. Strategy: Add inorganic phosphatase or use a continuous exchange cell-free system (dialysis-based CFPS).
  • Premature termination/Rare codons Strategy: Use extracts from optimized strains (like BL21 Rosetta) that overexpress tRNAs for rare codons.

Homework question from Kate Adamala

  1. Design an example of a useful synthetic minimal cell as follows: I) Pick a function and describe it. Biosensor for drug/contaminant degradation. a) What would your synthetic cell do? What is the input and what is the output? It would detect Doxycycline in the environment and emit a bioluminescence signal. Input: Doxycycline. Output: Light (Luciferase). b) Could this function be realized by cell-free Tx/Tl alone, without encapsulation? No, the response would be non-specific or low-sensitivity due to dilution. c) Could this function be realized by genetically modified natural cell? Yes, but the synthetic minimal cell is more modular and safer for controlled environments d) Describe the desired outcome of your synthetic cell operation. The desired outcome is a robust, switch-like biological response where the synthetic cell acts as a specific transducer. Upon exposure to the external target (Doxycycline), the synthetic cell must achieve the following:
  • Selective Sensing: The membrane-embedded OmpF channel must facilitate the passive diffusion of Doxycycline into the internal volume of the SMC without compromising the integrity of the lipid bilayer.
  • Transcriptional Activation: Once inside, Doxycycline must bind to the TetR repressor, inducing a conformational change that releases the operator site on the DNA template, allowing for the rapid synthesis of the reporter enzyme (e.g., Luciferase or GFP) via the encapsulated cell-free machinery.
  • Signal Amplification: The system must produce a sufficient concentration of the reporter protein to exceed the detection threshold of the measurement device (P51 viewer or luminometer) within a defined reaction window.
  • Defined Output: The final measurable state should be a binary “ON” signal (high fluorescence or bioluminescence) correlating directly to the presence of the input, while maintaining low background “OFF” signal in the absence of the target molecule.

II) Design all components that would need to be part of your synthetic cell. a) What would be the membrane made of? ​Phospholipids (POPC) + 10% Cholesterol (for mechanical stability). b) What would you encapsulate inside? Enzymes, small molecules. E. coli lysate, plasmid with Tet-ON promoter, luc gene (luciferase), and the OmpF membrane channel (pore for Doxycycline entry). c) Which organism your Tx/Tl system will come from? Is bacteria 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) System: E. coli (S30 extract). Bacterial is sufficient because the Tet-ON system is highly efficient in bacterial lysates. d) How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?) OmpF membrane channel (from E. coli). It allows for the selective entry of the antibiotic. III) 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.) luc (Luciferase from Photinus pyralis), tetR (Tet repressor), ompF. b) How will you measure the function of your system? ​Measurement: Plate reader luminometer (real-time photon emission measurement).

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:

  1. Write a one-sentence summary pitch sentence describing your concept. “Bio-Responsive Smart Insulation” is an architectural system utilizing freeze-dried, cell-free protein synthesis (CFPS) integrated into internal building insulation to detect and remediate structural moisture-induced microbial contamination through the colorimetric release of antimicrobial peptides.
  2. How will the idea work, in more detail? Write 3-4 sentences or more. The system consists of a fiber-based insulation mat embedded with freeze-dried CFPS pellets. In the event of a water leak or high-humidity breach in the building envelope, the moisture rehydrates the immobilized reaction components. This triggers the synthesis of a reporter protein for early detection (color change) and, subsequently, the expression of specific antimicrobial peptides (AMPs) or chitinases to inhibit fungal growth. By integrating the genetic circuitry directly into the building materials, the structure transitions from a passive object to an active, self-regulating biological system that prevents the degradation of interior structural integrity.
  3. What societal challenge or market need will this address? This addresses the massive global issue of “Sick Building Syndrome” caused by hidden mold growth in drywall and insulation. Beyond health benefits, it prevents costly, extensive structural repairs and reduces the environmental footprint of frequent building material replacement due to microbial rot.
  4. How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)? ​Activation: The system uses the moisture (leak/dampness) that it is designed to sense as the inherent trigger for activation. Stability: Freeze-dried pellets will be encapsulated in protective, breathable, semi-permeable polymers (like poly(vinyl alcohol) or silica-based aerogels) to prevent premature hydration while maintaining shelf-life. ​One-time use: Given the nature of structural leaks, the system acts as a “disposable fuse”; once activated to remediate a breach, the colorimetric change serves as a diagnostic marker for maintenance, signaling that the specific patch requires manual replacement.

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) Long-term space missions face the challenge of radiation-induced DNA damage to astronauts, which significantly increases cancer risk and genomic instability. Monitoring real-time DNA damage response (DDR) is crucial for human health in deep space. Current methods are limited by hardware weight and cold-chain requirements. A compact, cell-free diagnostic tool can provide rapid, actionable data on cellular stress levels without the need for living cell cultures, which are themselves highly sensitive to space radiation, offering a robust solution for astronaut health monitoring.
  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) The primary target is the expression of the p53 protein and downstream reporter genes (e.g., GFP or luciferase) controlled by p53-responsive DNA binding elements.
  3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words) p53 is the “guardian of the genome,” activated during DNA damage. By incorporating p53-responsive promoters into a BioBits® CFPS system, we can quantify the cellular response to cosmic radiation. If an astronaut’s blood or tissue sample contains high levels of DNA-damage-induced signaling molecules, the CFPS system will translate this into a measurable fluorescence signal via the P51 viewer. This links the molecular state of genomic stress directly to a visual output, allowing for real-time monitoring of radiation impact on the human body during deep-space transit.
  4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) Hypothesis: Freeze-dried BioBits® systems can be programmed to detect specific radiation-induced biomarkers in human physiological samples by coupling radiation-sensitive transcription factors to a synthetic gene circuit. Goal: To validate that a cell-free synthetic circuit can function reliably under microgravity conditions to detect DNA damage signals. The reasoning is that cell-free systems bypass the complexities of maintaining homeostatic viability in living cells under stress, providing a direct, quantitative measure of molecular signaling that is more robust and easier to interpret in the constrained resource environment of the International Space Station or future lunar habitats.
  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) We will test a synthetic circuit in a BioBits® reaction containing a plasmid with a p53-responsive promoter driving GFP. ​Samples: A “positive control” (synthetic biomarker mimic), a “negative control” (no biomarker), and irradiated vs. non-irradiated human blood-derived samples. ​Measurements: Use the miniPCR® to amplify DNA segments if necessary, and use the P51 Molecular Fluorescence Viewer to quantify GFP intensity. Data will be normalized against the non-irradiated samples to calculate a “Genomic Stress Index,” testing the system’s sensitivity to radiation-induced damage signatures in microgravity.