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.

In traditional in vivo (inside the cell) methods, the cell’s primary goal is its own survival. In CFPS, the goal is purely production.

  • Experimental Control: You can directly manipulate the environment. You can add non-natural amino acids, adjust redox potential, or add chaperones at precise concentrations without the cell’s homeostatic mechanisms fighting back.
  • Flexibility: There is no “transformation” step. You can add linear DNA or mRNA directly to the mix, enabling rapid prototyping (Design-Build-Test cycles).

Two Cases where CFPS is superior:

  • Cytotoxic Proteins: Producing proteins that would kill a living host (e.g., antimicrobial peptides or certain toxins) is much easier in vitro because the system doesn’t need to stay “alive.”
  • Incorporation of Non-Canonical Amino Acids (ncAAs): CFPS allows for the site-specific insertion of synthetic amino acids to create proteins with new chemical properties, which is often difficult in cells due to transport issues or toxicity.
  1. Describe the main components of a cell-free expression system and explain the role of each component.
  • Cell Extract (Crude Lysate): Provides the core machinery: Ribosomes, aminoacyl-tRNA synthetases, and initiation/elongation factors.
  • Cell-free protein synthesis template DNA or mRNA: Encodes the protein of interest.
  • Energy Source High-energy molecules (e.g., Phosphoenolpyruvate or Creatine Phosphate): to fuel the reaction.
  • Amino Acids: The raw building blocks for the protein chain.
  • Nucleotides (NTPs): Required for transcription (if starting from DNA) and energy transfer.
  • Salts and Buffers (e.g., Magnesium and Potassium): To maintain pH and stabilize the folding of the machinery.
  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 is energetically expensive. Every peptide bond requires the hydrolysis of multiple ATP and GTP molecules. If the energy supply is exhausted, translation stops, leading to low yields. One common method is the Secondary Energy Solution using Creatine Phosphate and Creatine Kinase. The kinase enzyme transfers a phosphate group from creatine phosphate to ADP, constantly “recharging” the ATP pool within the tube.

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

Prokaryotic (e.g., E. coli): High yield, fast, and inexpensive. However, it lacks post-translational modification (PTM) capabilities.

  • Protein to produce: Insulin. While complex, it is a small protein that can be efficiently produced and folded in optimized E. coli systems.

Eukaryotic (e.g., CHO or Wheat Germ): Lower yield but capable of complex folding and PTMs like glycosylation.

  • Protein to produce: Monoclonal Antibodies. These require complex disulfide bond formation and glycosylation to be functional, which prokaryotic systems cannot easily handle.
  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.

Membrane proteins are notoriously difficult because they are hydrophobic and require a lipid environment to fold correctly; otherwise, they aggregate and become useless.

Challenges and Solutions:

  • Challenge: Lack of a lipid bilayer.

  • Solution: Supplement the CFPS reaction with nanodiscs or synthetic liposomes. These provide a “landing pad” for the protein to insert itself into as it is being synthesized.

  • Challenge: Detergent toxicity.

  • Solution: Use detergent-free CFPS or mild surfactants that stabilize the protein without denaturing the translation machinery.

  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: Check the purity of your DNA/mRNA. Use RNase inhibitors to prevent the degradation of the blueprint during the reaction.
  • Energy Depletion: Analyze the duration of the reaction. If it stops prematurely, increase the concentration of the secondary energy source or implement a dialysis system to remove byproduct phosphate.
  • Poor Protein Folding: If the protein is being made but is insoluble (forming pellets), try lowering the incubation temperature or adding molecular chaperones (like DnaK/J) to the extract.

Homework question from Kate Adamala

1. Pick a Function and Describe It

1a. What would your synthetic cell do? What is the input and what is the output?

I would like to make a cell free system for diagnosis of Vibrio cholerae.

Function: Detect Vibrio cholerae presence in environmental samples (water, food, clinical samples) through recognition of cholera toxin A, and produce a visible/measurable output signal.

Input: Cholera Toxin A protein (CTA) or V. cholerae bacterial lysate containing CTA

Intermediate Process: CTA enters the SMC through a permeable membrane or receptor-mediated endocytosis-like mechanism. Inside the SMC, CTA is recognized by a detection system.

Output of SMC: Production of ADP-ribosylated protein (marking detection), followed by activation of a reporter gene

Output of whole system: Fluorescent protein (GFP) or enzymatic output (luciferase) proportional to CTA concentration

1b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

No.

  • Without encapsulation, the CTA would immediately diffuse away and not accumulate

  • There would be no way to separate the detection reaction from background noise in the environment

  • The synthetic cell’s advantage is creating a compartmentalized detection zone where high local concentrations of detection machinery exist

  • The membrane creates a controlled microenvironment where we can detect low concentrations of CTA that would be undetectable in bulk solution

  • Encapsulation allows us to recycle detection components and amplify the signal within the confined space

1c. Could this function be realized by genetically modified natural cell?

Yes, but with significant limitations:

Theoretically, I can transform E. coli or V. cholerae with genes for CTA-binding proteins and reporter genes

However, this approach has major problems:

  • Toxicity: CTA’s ADP-ribosylation activity could damage the host cell’s essential proteins, killing the cell or triggering SOS response
  • Biocontainment: Releasing genetically modified bacteria for field diagnostics is problematic (regulatory, safety, environmental concerns)
  • Specificity: Natural cells have their own metabolic activities that interfere with clean detection
  • Standardization: Cell-free systems are more reproducible and easier to quality-control than living cells
  • Cost: A synthetic cell biosensor is cheaper to produce and deploy than maintaining living cell cultures

We avoid the toxicity problem because cell-free translation doesn’t depend on maintaining cellular homeostasis or viability.

1d. Describe the desired outcome of your synthetic cell operation.

Desired Outcome: When a water sample (potentially contaminated with V. cholerae) is added to the SMC suspension:

  • Detection Phase (0-5 minutes): CTA molecules from the sample cross the SMC membrane and encounter our detection machinery inside
  • Signal Amplification (5-30 minutes): Positive detection triggers transcription/translation of GFP reporter gene
  • Output (30+ minutes): Fluorescence increases proportionally to CTA concentration
  • Readout: Using a simple fluorimeter (or even a smartphone with UV light), the presence of V. cholerae is confirmed - no bacterial culture needed, results in <1 hour

Clinical Application: A field-deployable diagnostic tool for cholera detection in resource-limited settings.

2. Design All Components of the Synthetic Cell

2a. What would be the membrane made of?

Membrane Composition:

POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine): 60-70% → Main phospholipid providing bilayer structure → Chosen because it’s fluid at room temperature and biologically relevant

Cholesterol: 20-25% → Increases membrane mechanical stability → Reduces permeability to small hydrophilic molecules we want to control → Improves long-term storage stability of vesicles

DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]): 5-10% → Provides stealth properties (reduces non-specific protein absorption) → Improves biocompatibility if used in human samples → Prevents aggregation of SMC vesicles with each other

Optional: Sphingomyelin (5-10%) → Increases membrane rigidity and lifespan → Creates more ordered membrane domains

  • This formulation is based on successful demonstrations and mimics the complexity of biological membranes
  • The POPC:cholesterol ratio (60:25) provides optimal fluidity while maintaining stability
  • We keep it simple enough to be reproducible but complex enough to be functional

2b. What would you encapsulate inside? Enzymes, small molecules.

Encapsulated Components:

A. Cell-Free Translation/Transcription System (Tx/Tl):

  • Complete bacterial extract (PURE system or S30 extract from E. coli)
  • NTPs (ATP, GTP, CTP, UTP) - 2-4 mM each
  • Amino acids (all 20 standard ones) - 200-500 µM each
  • Energy regeneration system: PEP (phosphoenolpyruvate) and PEP synthetase
  • Ribosomes, tRNAs, various factors for transcription and translation

B. Detection Cassette (encoded in plasmid DNA):

  • CTA-Binding Domain: Nanobody (single-domain antibody) against cholera toxin A, expressed as a fusion protein

Nanobodies are smaller than full antibodies, easier to express in cell-free systems

  • Signal Transduction: Upon CTA binding to our detection protein, we trigger transcription of reporter gene

Use a constitutive T7 promoter (CTA presence alone triggers transcription)

C. Reporter Gene (in plasmid or added separately):

GFP (green fluorescent protein) gene under constitutive T7 promoter

Output: Green fluorescence at 509 nm

Easy to measure with standard equipment, visible signal

D. Supporting Small Molecules:

  • Magnesium chloride (MgCl₂): 5-10 mM (cofactor for ribosomes and polymerases)
  • Potassium glutamate: 100-150 mM (osmolyte, maintains ionic balance)
  • DTT (dithiothreitol): 1-2 mM (reduces oxidative stress, keeps proteins in reduced state)
  • EDTA: 0.1-0.5 mM (chelates metal contaminants)

E. DNA Encoding the Detection Circuit (Plasmid DNA):

Gene 1: Nanobody against CTA (under T7 promoter) Gene 2: GFP reporter (under constitutive T7 promoter)

2c. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system?

Bacterial (E. coli) is appropriate and optimal here. It costs less to use a bacterial system, more stable and robust, can incooperate the T7 promoter mechanism, and there is no need for post translation modification.

2d. How will your synthetic cell communicate with the environment?

Passive Diffusion

CTA is a ~25 kDa protein - too large to freely diffuse across lipid bilayer. However the following can be done to ensure that their is diffusion

  • Make the membrane slightly permeable to proteins
  • Use higher cholesterol content (to 30-35%) to increase membrane fluidity, this allows some leakage of proteins across the membrane

3. Experimental Details

3a. List all lipids and genes.

  • Lipids: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), Cholesterol, GM1 Ganglioside (Cholera Toxin receptor)
  • Proteins and enzymes: T7 RNA Polymerase, E. coli ribosomes
  • Gene Construct 1: CTA Detection & Signal Transduction
  • small molecules: ATP,GTP, CTP, UPT, All 20 amino acids

3b. How will you measure the function of your system?

Fluorescence-based detection

Homework question from Ally Huang

(Queried ChtaGpt, Gemini and Claude on this to see what the best approach would be for solving DNA damage, with a bias to rapid diagnostics as this is an area i am interested in)

  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-duration space missions expose astronauts to cosmic radiation, causing DNA damage and muscle/bone loss. Preserving astronaut health is critical, but traditional medical diagnostic equipment is too heavy and resource-intensive for spacecraft. We need lightweight, on-demand tools to detect cellular stress early. Solving this is significant for humanity’s future as a multiplanetary species, relevant for safe Mars exploration, and scientifically interesting because it tests how synthetic biology can replace massive labs with paper-based, freeze-dried cellular sensors.

  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)

The p53 tumor suppressor protein. It is known as the “guardian of the genome” and is rapidly produced by human cells in response to radiation-induced DNA damage.

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

When astronauts are exposed to harmful space radiation, their cells experience DNA double-strand breaks. This damage activates the p53 pathway to either repair the cell or trigger cell death. By targeting the expression of the p53 gene, we can create a direct, biological warning system. If an astronaut’s blood or saliva sample triggers high p53 activity, it indicates dangerous levels of radiation exposure. Monitoring this target allows real-time health tracking before severe symptoms, like cancer or radiation sickness, physically manifest during a mission.

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

Freeze-dried BioBits® cell-free reactions can be successfully engineered to produce a visible green fluorescent protein (GFP) when activated by synthetic DNA plasmids mimicking astronaut p53 expression.

BioBits® contains all the cellular machinery (ribosomes, enzymes) needed to translate DNA into proteins without needing live cells. In space, live cell cultures easily die and are hard to maintain. Freeze-dried BioBits® are shelf-stable, lightweight, and activate simply by adding water. By linking a p53-responsive promoter sequence to a GFP gene, any p53 activation will trigger GFP production. This will cause the sample to glow brightly under the P51 Molecular Fluorescence Viewer, proving that cell-free systems can reliably diagnose radiation stress without requiring a traditional, heavy laboratory footprint.

  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)

Synthetic DNA templates representing astronaut samples under radiation stress will be tested.

  • Experimental Sample: BioBits® rehydrated with DNA containing the p53-activated GFP gene.
  • Positive Control: BioBits® with a standard, always-active GFP control DNA (to ensure the kit works).
  • Negative Control: BioBits® rehydrated with sterile water (no DNA) to check for background glow.
  • Samples will be incubated in the miniPCR® thermal cycler to mimic body temperature.
  • Data will be collected by placing the tubes into the P51 Fluorescence Viewer and measuring visual fluorescence intensity (glowing vs. dark).

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. A paper-based, low-cost diagnostic tool that utilizes freeze-dried cell-free systems and RNA toehold switches to provide a visible color-change signal in the presence of Vibrio cholerae within 60 minutes.

  • How will the idea work, in more detail? Write 3-4 sentences or more. The device consists of a paper strip embedded with a freeze-dried cell-free transcription-translation (TX-TL) system and a synthetic “toehold switch” gene circuit. When a suspected water sample is dropped onto the paper, the water rehydrates the biological machinery, “waking it up.” If the specific RNA sequence of the Vibrio cholerae bacteria is present in the sample, it binds to the toehold switch, unfolding the RNA structure and allowing the ribosome to translate a reporter protein, such as LacZ or purple chromoprotein, which turns the paper from white to a distinct color.

  • What societal challenge or market need will this address? Cholera remains a major global health threat in resource-limited settings where traditional lab-based testing (like bacterial culture or PCR) is too slow, expensive, and dependent on a “cold chain” for refrigerated transport. This kit addresses the need for decentralized, rapid outbreak detection, allowing local health workers to confirm cases in the field and immediately initiate life-saving interventions like water chlorination and rehydration therapy.

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

Activation: The limitation of needing water for activation is turned into a benefit here, as the liquid sample being tested serves as the rehydration agent for the freeze-dried reagents.

Stability: By incorporating lyoprotectants (such as trehalose) during the freeze-drying process, the enzymatic machinery is stabilized against high tropical temperatures, eliminating the need for refrigeration during shipping.

One-Time Use: To ensure the tool is economically viable despite being one-time use, the system is engineered onto cellulose paper, which is biodegradable and costs only cents per test, making it a scalable solution for mass surveillance during floods or humanitarian crises.