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.

Flexibility:

• All components (DNA template, ribosomes, substrates, cofactors, chaperones, detergents) can be added, removed, or tuned in real time without the constraints of cellular membranes or homeostasis.
• No need for cloning, transformation, culture optimization, or induction. Protein production begins within minutes to hours.
• Proteins that are lethal to cells, misfolded in vivo, or subject to proteolytic degradation can be synthesized without harming host viability.

Control over experimental variables:

• can be precisely set and maintained.
• easier than metabolic incorporation; simply add ncAA and orthogonal tRNA/synthetase.
• ATP/GTP levels and redox balance (NAD⁺/NADH) can be independently controlled.

Two cases where cell-free is more beneficial:

1: Production of toxic or membrane-disruptive proteins. These proteins lyse or kill host cells, preventing scalable in vivo production. Cell-free systems bypass cell viability entirely.

2: Incorporation of multiple non-canonical amino acids for structural/biophysical studies. Metabolic incorporation in vivo is limited by cellular uptake, toxicity, and competition with natural amino acids. Cell-free systems allow direct supplementation of ncAAs at high concentrations with orthogonal suppressor tRNAs, achieving near-complete incorporation at desired positions.

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

A typical prokaryotic cell-free system (e.g., E. coli S30 or PURE system) contains:

3. Why is energy provision and regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.

• ATP and GTP are rapidly consumed during transcription (NTP incorporation), translation (aminoacyl-tRNA charging, peptide bond formation, ribosome translocation), and chaperone activity.
• Unlike living cells, cell-free systems lack glycolysis and oxidative phosphorylation and cannot regenerate ATP metabolically.
• Without regeneration, ATP is depleted within minutes, halting protein synthesis prematurely and drastically reducing yield.

Method to ensure continuous ATP supply:

Phosphoenolpyruvate (PEP) / pyruvate kinase system:

• Mechanism:
  PEP + ADP →^(pyruvate kinase) ATP + pyruvate
• Advantages:
  • PEP has a very high phosphoryl transfer potential (ΔG°′ ≈ –62 kJ/mol), driving ATP regeneration efficiently.
  • Pyruvate is an end product and does not inhibit the reaction.
  • Simple to implement; widely used in commercial kits.
• Procedure:
  1. Add PEP (e.g., 20–50 mM) and pyruvate kinase (e.g., 0.1–0.5 U/μL) to the reaction mix.
  2. Monitor reaction time; PEP-based systems can sustain synthesis for 2–6 hours or longer.

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

Protein choice for prokaryotic system:

Protein: Cas9 endonuclease (e.g., Streptococcus pyogenes Cas9)

• Cas9 is a bacterial protein with no requirement for glycosylation or complex eukaryotic PTMs.
• High yield is desirable for genome editing applications and structural studies.
• E. coli cell-free systems (especially PURE or S30 extracts) produce functional Cas9 rapidly and cost-effectively.
• Can incorporate ncAAs for labeling or crosslinking studies without eukaryotic metabolism interference.

Protein choice for eukaryotic system:

Protein: Erythropoietin (EPO) – a therapeutic glycoprotein hormone

• EPO requires N-linked glycosylation for stability, serum half-life, and biological activity.
• Prokaryotic systems cannot perform glycosylation; EPO produced in E. coli is inactive or rapidly degraded in vivo.
• Eukaryotic cell-free systems (e.g., CHO lysate, insect cell extract, or wheat germ supplemented with microsomes) can glycosylate nascent chains co-translationally or post-translationally.
• Cell-free allows rapid prototyping of glycosylation site mutants without lengthy cell-line development.

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.

Challenges of membrane protein expression in cell-free systems:

1. Hydrophobicity → aggregation: Membrane proteins have large hydrophobic transmembrane domains that aggregate and precipitate in aqueous solution.
2. Loss of native structure and function: Without a lipid bilayer, transmembrane proteins misfold.
3. Low solubility and yield.

Experimental design to optimize membrane protein expression:

1. Choice of membrane-mimetic environment:

Detergent micelles

• Add nonionic detergents (e.g., Brij-35, digitonin, DDM) or zwitterionic detergents (Fos-Choline) directly to the reaction.
• Detergents solubilize hydrophobic domains co-translationally, preventing aggregation.
• Optimization: titrate detergent concentration (typically 0.5–2% w/v) to balance solubilization vs ribosome inhibition.

2. Use of chaperones and lipid-transfer machinery:

• Supplement with YidC (bacterial insertase), Sec translocon components, or eukaryotic SRP (signal recognition particle) if using eukaryotic lysates.
• Enhance folding with molecular chaperones (e.g., DnaK, GroEL/ES).

3. Template design:

• Use constructs with solubility tags (e.g., MBP, SUMO) fused to membrane protein to aid initial solubility, followed by protease cleavage.
• Optimize the 5′ UTR and RBS/Kozak sequence for efficient translation initiation.

4. Optimization strategy:

• Small-scale screening: vary detergent type/concentration, liposome composition, Mg²⁺, K⁺, temperature (often lower temps ~25–30°C improve folding).
• Functional assay readout: ligand binding, fluorescence (if

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.

Reason 1: Inefficient transcription or translation initiation

Troubleshooting strategy:

• Check DNA template integrity: run agarose gel to confirm template is intact and at expected size.
• Verify transcription: isolate RNA from a small aliquot of the reaction and run on denaturing gel or use RT-qPCR to quantify mRNA levels. Low mRNA → transcription problem.
• Optimize RBS/5′ UTR: test different spacing between RBS and start codon (optimal is ~5–9 nt for E. coli); use strong synthetic RBS sequences (e.g., BBa_B0034).
• Add more T7 RNA polymerase or fresh enzyme if transcription is limiting.

Reason 2: Protein aggregation or misfolding

Troubleshooting strategy:

• Add molecular chaperones: supplement with DnaK/DnaJ/GrpE (Hsp70 system), GroEL/ES, or trigger factor.
• Adjust redox conditions: for proteins requiring disulfide bonds, add oxidized glutathione (GSSG) or protein disulfide isomerase (PDI). For reducing environment, add DTT or β-mercaptoethanol.
• Lower reaction temperature: reduce from 37°C to 25–30°C to slow synthesis and allow more time for folding.
• Check solubility: centrifuge reaction and analyze supernatant vs pellet by SDS-PAGE. If protein is in pellet, it aggregated → try detergents or co-expression of solubility tags (MBP, SUMO).

Reason 3: Rapid degradation of the target protein

Troubleshooting strategy:

• Add protease inhibitors: include a cocktail (e.g., PMSF, leupeptin, pepstatin, aprotinin, or EDTA-free complete protease inhibitor tablets).
• Use nuclease-treated lysates: commercial kits often pre-treat extracts to remove nucleases and reduce protease activity.
• Time-course experiment: sample the reaction at multiple time points (e.g., 30 min, 1 h, 2 h, 4 h) and run Western blot or SDS-PAGE. If protein appears early then disappears → degradation. If it never appears → synthesis problem.
• Stabilize protein: add ligands, substrates, or cofactors that stabilize the native fold.
• Check sequence: ensure no internal stop codons, frameshifts, or rare codons that stall ribosomes (leading to abortive peptides).

Homework question from Kate Adamala

1. Pick a function and describe it

a1. What would your synthetic cell do?

The synthetic minimal cell (SMC) will function as a heavy metal biosensor and detoxification system. It will detect toxic mercury ions (Hg²⁺) in water and respond by producing a fluorescent signal and binding the mercury ions to reduce toxicity.

a2. What is the input and output?

• Input: Mercury ions (Hg²⁺)
• Output:
  1. Green fluorescent protein (GFP) signal
  2. Mercury-binding protein (MerP)

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

Partially, yes. A cell-free Tx/Tl system could detect Hg²⁺ and produce GFP in solution. However, encapsulation is important because:

• it protects the reaction components,
• increases system stability,
• prevents diffusion of proteins,
• allows controlled interaction with the environment.

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

Yes. Genetically modified E. coli could perform the same task using the mercury resistance operon. However, synthetic minimal cells are safer because:

• they are non-living,
• they cannot replicate,
• they reduce biosafety concerns,
• they are easier to control experimentally.

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

When mercury ions are present:

1. Hg²⁺ enters the synthetic cell.
2. A mercury-responsive genetic circuit activates.
3. GFP fluorescence is produced.
4. MerP binds mercury ions.
5. The contaminated sample becomes fluorescent and partially detoxified.

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

a. What would the membrane be made of?

The membrane will consist of:

• POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)
• Cholesterol
• DSPE-PEG2000 for additional membrane stability

Example membrane composition:

• 70% POPC
• 25% cholesterol
• 5% DSPE-PEG2000

b. What would you encapsulate inside?

Cell-free Tx/Tl system

A bacterial transcription/translation system containing:

• ribosomes,
• tRNAs,
• amino acids,
• RNA polymerase,
• ATP regeneration system,
• nucleotides,
• cofactors.

Small molecules

• ATP
• GTP
• amino acids
• Mg²⁺ ions
• K⁺ buffer
• phosphoenolpyruvate (energy source)

DNA plasmids

Plasmids encoding:

1. Mercury-responsive regulator (merR)
2. Green fluorescent protein (gfp)
3. Mercury-binding protein (merP)
4. Membrane pore protein (hla)

c. Which organism will the Tx/Tl system come from?

The Tx/Tl system will come from bacterial E. coli extract. A bacterial system is sufficient because:

• mercury-responsive promoters naturally exist in bacteria,
• bacterial Tx/Tl systems are inexpensive and robust,
• no mammalian-specific regulation is required.

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

Mercury ions are small enough to diffuse slowly through lipid membranes. To improve transport efficiency, the membrane will contain pores formed by α-hemolysin.

Communication mechanism

1. Hg²⁺ enters through α-hemolysin pores.
2. Internal genetic circuit activates.
3. GFP accumulates inside the vesicle.
4. Fluorescence can be measured externally.

3. Experimental detail

a. List all lipids and genes

Lipids: POPC, Cholesterol, DSPE-PEG2000 Genes: merR, merP, gfp, hla

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

Measure GFP fluorescence using fluorescence microscopy, microplate reader, flow cytometry. Measure mercury removal using atomic absorption spectroscopy (AAS), ICP-MS (inductively coupled plasma mass spectrometry).

Homework question from Peter Nguyen

One-Sentence Pitch

Freeze-dried cell-free biosensor systems embedded in façade panels enable buildings to detect airborne pollutants and trigger visible color changes or catalytic neutralization in response to poor air quality.

How the Idea Works

The concept integrates freeze-dried, cell-free synthetic biology systems directly into porous architectural materials such as façade tiles, wall panels, or concrete coatings. These systems contain DNA-encoded biosensors that respond to specific airborne pollutants (e.g., NOx, SO₂, formaldehyde, or particulate-associated toxins).

Upon activation by environmental moisture (rain, humidity, or condensation), the cell-free reaction initiates transcription–translation processes that produce either:

1. A visible pigment (e.g., chromoprotein) that changes the color of the surface to signal pollution levels, and/or
2. An inducible enzyme (e.g., oxidoreductases or peroxidases) capable of catalytically degrading certain pollutants at the material surface.

The biological components are compartmentalized in microcapsules distributed throughout the material matrix. When exposed to specific chemical triggers, the capsules activate locally, enabling spatially resolved sensing across the building envelope.

Societal Challenge / Market Need

Urban air pollution is a major public health issue linked to respiratory and cardiovascular diseases. However, pollution monitoring infrastructure is sparse, expensive, and often spatially limited. Buildings themselves represent large, underutilized surface areas in cities.

This system transforms passive architectural surfaces into distributed environmental sensors and potentially active remediation platforms. It addresses the need for:

• Hyperlocal, real-time pollution monitoring
• Increased public awareness of environmental conditions
• Integration of sustainability and smart functionality into construction materials

Addressing Limitations of Cell-Free Systems

1. Water Activation Freeze-dried systems are naturally stable in dry conditions and activate upon hydration. In architecture, this can be leveraged by designing materials that respond specifically to rain events or high humidity, which often correlate with pollutant deposition.

2. Stability The cell-free components can be stabilized using:

• Trehalose or other lyoprotectants
• Polymer encapsulation
• UV-protective coatings
• Encapsulation in hydrogel microdomains within the material can further enhance thermal and environmental stability.

3. One-Time Use Limitation

• The material can contain distributed micro-reservoirs that activate sequentially over time.
• Replaceable surface coatings or modular façade tiles can be designed for periodic renewal.
• Alternatively, the system can be designed as an event-reporting sensor (e.g., long-term color change after threshold exposure), functioning as a cumulative environmental record rather than a continuously active device.

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.

Long-duration spaceflight exposes astronauts to elevated levels of ionizing radiation, increasing the risk of DNA damage, cancer, and degenerative diseases. Current monitoring systems primarily measure physical radiation dose rather than biological impact. A lightweight, low-resource method to assess functional DNA damage in space would improve astronaut health monitoring and countermeasure development. Cell-free systems are particularly suitable for space missions due to their stability, low mass, and independence from living cells. Developing a molecular assay that links radiation exposure to functional gene expression capacity addresses a critical challenge for deep-space exploration missions to the Moon and Mars.

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.

Radiation-induced DNA double-strand breaks in a GFP reporter plasmid, quantified through reduction of functional protein expression in the BioBits® cell-free system.

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

Ionizing radiation induces DNA double-strand breaks (DSBs), among the most severe forms of genomic damage. DSBs disrupt gene integrity and impair transcription and translation. A plasmid encoding GFP can serve as a functional reporter: radiation-induced strand breaks decrease or abolish GFP expression in a cell-free system. Therefore, fluorescence intensity directly reflects template integrity. This strategy measures biologically meaningful damage rather than only absorbed radiation dose. By linking molecular lesions to protein synthesis capacity, the assay provides insight into how radiation may compromise essential genetic processes during spaceflight.

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

Space radiation exposure reduces functional GFP expression from a reporter plasmid in the BioBits® cell-free system in proportion to accumulated DNA damage.

Ionizing radiation generates strand breaks and base modifications that interfere with transcription and translation. If a GFP-encoding plasmid is exposed to spaceflight conditions and subsequently used in a cell-free protein expression reaction, DNA lesions should decrease protein yield. By comparing fluorescence output between spaceflight-exposed and Earth-based control plasmids, we can quantify functional DNA integrity. This approach provides a biologically relevant readout of radiation damage using minimal hardware. The research goal is to validate a compact, rapid assay capable of assessing molecular radiation damage during long-duration space missions.

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.

Identical GFP plasmid samples will be divided into: (1) spaceflight-exposed samples and (2) ground-based controls. After exposure, plasmids may be amplified using the miniPCR® (to assess amplifiability) and then expressed using the BioBits® cell-free protein expression system. GFP fluorescence will be measured with the P51 Molecular Fluorescence Viewer. Controls include no-DNA reactions (negative control) and non-exposed plasmid reactions (positive control). Fluorescence intensity will be quantified and normalized to DNA input. Reduced fluorescence in spaceflight samples relative to controls will indicate functional radiation-induced DNA damage.

Homework Part B: Individual Final Project