Week 9

Class Assignment — Week 9

Part A. General and Lecturer-Specific Questions

1. General homework questions

1. Advantages of Cell-Free Protein Synthesis Over In Vivo Methods

Cell-free systems decouple protein production from cell viability, giving you direct control over reaction composition, temperature, redox state, and cofactor concentrations, none of which are easily tunable in living cells.

Two cases where CFPS outperforms cell-based production:

• Viral biosensors / NTDs: Rapid, open-system format allows same-day prototyping of diagnostic reagents without biosafety constraints of live pathogen handling.
• Accessible diagnostic biomarkers (e.g., creatinine sensors for CKD): Low-cost E. coli extracts enable point-of-care biosensor manufacturing without fermentation infrastructure.

2. Main Components of a Cell-Free Expression System

3. Why Energy Regeneration Is Critical in Cell-Free Systems

Without regeneration, ATP is exhausted within minutes, translation stalls before any useful yield accumulates.

Method — Phosphoenolpyruvate (PEP) Regeneration:

• PEP donates a phosphate group to ADP via pyruvate kinase, regenerating ATP continuously throughout the reaction.
• It is the most widely used system in E. coli-based CFPS; simple to implement and well-characterised.

Alternatives:

• Glucose-6-phosphate / glycolysis: Cost-effective; couples to endogenous glycolytic enzymes in the extract.
• Creatine phosphate / creatine kinase: Common in eukaryotic systems; mimics the muscle energy buffering mechanism.

4. Prokaryotic vs. Eukaryotic Cell-Free Expression Systems

Protein choice — Prokaryotic: GFP

• GFP is small, soluble, and folds spontaneously without PTMs — perfect for E. coli CFPS.
• Fluorescence output doubles as a real-time yield reporter; ideal for rapid system validation.
• High-throughput expression kits for GFP are cheap, reproducible, and produce results in under 4 hours.

Protein choice — Eukaryotic (CHO/HeLa): IgG Monoclonal Antibody

• IgG requires N-glycosylation, disulfide bond formation, and ER-assisted folding for activity.
• CHO/HeLa lysates contain ER-derived microsomes with glycosylation enzymes and PDI — E. coli cannot replicate this.
• Attempting IgG expression in prokaryotic CFPS typically yields insoluble, non-functional aggregates.

5. Designing a Cell-Free Experiment for Membrane Protein Expression

Membrane proteins (MPs) are notoriously difficult — aggregation, low yield, and incorrect insertion are the default failure modes. My approach centres on a Continuous Exchange Cell-Free (CECF) setup with deliberate hydrophobic stabilisation from the moment of synthesis.

Experimental Design:

• Template: PCR-derived linear DNA with T7 promoter; codon-optimised for the chosen lysate; RBS positioned ~11 nt upstream of ATG.
• Chassis: E. coli extract for yield; insect or HeLa lysate if the MP needs native PTMs or microsomal insertion.
• Hydrophobic additives: Supplement with detergents (Brij-35, LMNG) or nanodiscs directly in the reaction to catch the MP co-translationally.
• CECF mode: Use a 10× feeding solution volume to replenish ATP, amino acids, and dilute inhibitory byproducts over 4–16 hours.
• Temperature: Start at 25–30 °C to slow translation and reduce aggregation kinetics.

Challenges and Solutions:

• Aggregation: Add nanodiscs or lipid vesicles to provide a bilayer scaffold immediately upon synthesis.
• mRNA/DNA degradation: Use GamS protein to block RecBCD exonuclease activity on linear templates.
• Incorrect folding: Introduce pre-formed inverted membrane vesicles or switch to insect lysate with native microsomes.
• Codon bias (eukaryotic MP in E. coli): Codon-optimise the sequence or switch to wheat germ / rabbit reticulocyte lysate.
• Low-throughput screening: Miniaturise to microfluidic volumes; automate condition matrices varying detergent type and temperature.

6. Troubleshooting Low Yield in a Cell-Free System

Reason 1 — Protein Aggregation / Misfolding:

• Misfolded hydrophobic stretches form inclusion bodies, reducing soluble yield.
• Fix: Drop incubation temperature to 25 °C to slow translation and buy time for folding.
• Fix: Add solubility tags (Mocr, GST) or co-express chaperones (DnaK/DnaJ/GrpE) in the reaction.

Reason 2 — Premature Energy Depletion:

• PEP or creatine phosphate runs out before the reaction plateau, stalling ribosomes mid-synthesis.
• Fix: Switch to a CECF dialysis setup to continuously feed energy substrates and remove Pi accumulation.
• Fix: Supplement with additional glucose as a secondary energy source to extend reaction lifetime.

Reason 3 — Low Transcription / Translation Efficiency:

• Weak promoter, suboptimal DNA concentration, or mRNA degradation by endogenous RNases.
• Fix: Optimise plasmid concentration (typically 5–20 nM); confirm strong T7 promoter; add RNase inhibitor (e.g., RiboLock).
• Fix: Verify T7 RNA polymerase activity separately; use circular plasmid rather than linear DNA if exonuclease degradation is suspected.

2. Homework question from Kate Adamala

Overview

The Synthetic Neuronal Mimic (SNM) is a liposome-based minimal cell designed as an interactive, safe, and visual educational tool for youth STEM leaders to understand the impact of drugs on biological systems.

1. Function Description

a. What does the SNM do? What is the input and output?

• Function: The SNM acts as a miniature “biological laboratory” encapsulating a cell-free TX/TL system that produces a fluorescent signal only when a specific drug molecule is present.
• Input: A drug molecule (e.g. nicotine analog, stimulant) in the surrounding environment, which diffuses through the synthetic membrane via a pore channel.
• Output: sfGFP fluorescence, visible under a portable fluorescence microscope. Signal intensity is a direct visual proxy for drug dose or effect magnitude.

b. Could cell-free TX/TL alone, without encapsulation, realise this function?

• No. TX/TL in a tube produces the protein but loses the educational purpose entirely.
• Encapsulation creates a compartmentalised entity that behaves like a cell, not a chemical mix.
• The drug must cross a synthetic membrane before the circuit responds, directly mirroring how neurons work.
• Without encapsulation, you have chemistry. With it, you have a cell.

c. Could a genetically modified natural cell realise this function?

• Yes, but it is the wrong tool for this context.
• Engineered E. coli or yeast would require biosafety containment, specialised culture media, and are prone to mutation.
• The SNM contains no living organism, making it safer to handle in outreach settings.
• It is more predictable, easier to explain from first principles, and requires no microbiology infrastructure.

d. Desired outcome of SNM operation

• Youth STEM leaders directly observe drug-responsive circuit logic in real time.
• Input A (nicotine analog) produces Output B (high-intensity GFP fluorescence).
• Participants leave with a concrete, visual understanding of how microscopic chemical signals produce measurable biological responses.
• The experience serves as a practical entry point into pharmacology and neuroscience.

2. Component Design

a. Membrane composition

• Phospholipid bilayer: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and cholesterol at an 80:20 molar ratio.
• Cholesterol increases membrane rigidity and reduces passive leakage of internal components.
• Alpha-hemolysin (alpha-HL, gene: hla) is embedded in the bilayer to create ~2 nm pores that admit small molecules up to ~2 kDa.

b. Internal encapsulation

• E. coli S30 or PUREsystem cell-free extract: supplies ribosomes, RNA polymerase, tRNA, and chaperones.
• Plasmid encoding sfGFP under a TetR-repressible promoter (pTet).
• ATP, GTP, and a full complement of amino acids.
• PEP-based ATP regeneration system (phosphoenolpyruvate + pyruvate kinase).
• RNase inhibitor (e.g. RiboLock) to protect mRNA from endogenous nuclease activity.

c. TX/TL system origin: bacterial or mammalian?

• Bacterial (E. coli) extract is sufficient for this design.
• TetR/pTet is fully functional in prokaryotic cell-free systems; no mammalian system is required.
• E. coli extract is low-cost, freeze-dryable for outreach kit distribution, and yields high sfGFP concentrations within 2 to 4 hours.
• A mammalian system would only be necessary if the circuit required PTMs or mammalian-specific promoter logic, which this design does not.

d. Communication with the environment

• The SNM communicates via passive diffusion through alpha-HL pores.
• The drug analog (small molecule, up to ~2 kDa) enters through the pore and de-represses the TetR-controlled sfGFP promoter.
• No active transport machinery or membrane receptors are required.

3. Experimental Details

a. Lipids and genes

b. Measuring system function

• Primary readout: Fluorescence microscopy using a portable LED scope (470 nm excitation / 510 nm emission); visible GFP signal confirms circuit activation.
• Quantification: Plate reader measuring fluorescence intensity (Ex 485 nm / Em 510 nm) as a function of drug concentration to generate a dose-response curve.
• Negative control: SNMs incubated without drug input; no fluorescence expected, confirming the circuit is OFF at baseline.
• Positive control: SNMs with a constitutive always-on sfGFP construct; calibrates maximum signal and confirms TX/TL machinery is functional.
• Validation metric: Signal-to-noise ratio of drug-treated vs. no-drug control; a minimum 5-fold induction threshold confirms adequate circuit sensitivity.

3. Homework question from Peter Nguyen

Application Field

Architecture — wellness-focused interior design using nature-based, intelligent building materials.

One-Sentence Pitch

The Neuro-BioWall is a modular interior wall panel system embedding freeze-dried cell-free biosensors within living plant scaffolds to detect indoor air pollutants and respond with enzyme-triggered aromatherapy, bridging passive biophilic design and active biological intelligence.

How It Works

The system consists of 3D-printed cellulose/alginate panels hosting living Pothos plants, with freeze-dried cell-free reactions integrated directly into the plant’s nutrient-delivery interface. When indoor VOCs such as formaldehyde exceed healthy thresholds, a toehold switch genetic circuit embedded in the cell-free system is activated, initiating synthesis of a reporter enzyme. That enzyme acts on a co-encapsulated, latent aromatherapeutic substrate to release a localised calming scent such as lavender or hinoki. Simultaneously, a colorimetric output produces a visible colour change in the biopolymer panel, giving occupants a passive, non-electronic visual cue to ventilate or pause.

Step-by-step workflow:

1. Pollutant intake: Indoor air flows through the porous biocellulose pot interface where plant roots and cell-free sensors reside.
2. Sensing: The cell-free toehold switch circuit triggers when VOC concentrations exceed the design threshold.
3. Wellness output: The activated circuit produces an esterase enzyme that breaks down a sealed aromatherapeutic compound, releasing scent.
4. Visual signal: Colorimetric reporter causes a visible change in the biopolymer scaffold, prompting occupants to take action.

Societal Challenge and Market Need

• Sick building syndrome affects an estimated 30% of office buildings globally, linked to VOC accumulation from furniture, adhesives, and cleaning products.
• Existing solutions are either passive (plants, carbon filters) with no active feedback, or electronic (air quality monitors) with no biological or sensory integration.
• The Neuro-BioWall closes this gap: it monitors, responds, and communicates without electronics, live microbes, or occupant intervention.
• It targets the growing wellness architecture and biophilic design market, where demand for nature-integrated, low-maintenance intelligent building materials is expanding rapidly.

Addressing Cell-Free System Limitations

Activation with water

• The cell-free components are freeze-dried directly into the hydrogel of the plant nutrient scaffold.
• Activation occurs automatically during the plant’s regular watering cycle, requiring no separate triggering step or electronic control.

Long-term stability

• Components are lyophilised in a trehalose-based sugar matrix and encapsulated within a protective polymer mesh.
• This configuration maintains activity at room temperature for 3 to 6 months without refrigeration.
• The trehalose matrix is a well-established stabilisation strategy for cell-free systems in low-resource and distributed deployment contexts.

One-time use

• The sensor is packaged as a replaceable modular bio-cartridge that clips in and out of the living panel.
• Spent cartridges are fully biodegradable, consistent with the cellulose/alginate material system.
• Routine cartridge replacement is designed as a simple maintenance step, analogous to changing a water filter, rather than a structural intervention.

Integrated Material Summary

Why This Works as a Platform

• No living microbes means no biosafety concerns in occupied buildings.
• No electronics means no power dependency, no failure modes from software or connectivity.
• The plant’s natural water cycle doubles as the activation mechanism, making the system self-sustaining within normal building maintenance routines.
• Modular cartridge design allows iterative sensor upgrades without replacing the structural panel, extending product lifetime and reducing material waste.

4. Homework question from Ally Huang

Overview

MycoLab-1 proposes a minimally functional, university-grade biological sciences laboratory for deep-space environments, built from mycelium-based composite (MBC) infrastructure and powered by freeze-dried cell-free (CFPS) molecular biology systems. The laboratory requires no refrigeration chain, no live microbial culture infrastructure, and no heavy equipment payload — making advanced biological experimentation feasible aboard lunar outposts, Mars transit vehicles, or orbital stations where mass and power budgets are severe constraints.

1. Background: The Space Biology Challenge

Long-duration spaceflight exposes crew to ionising radiation, microgravity-induced immune dysregulation, and chronic oxidative stress — all of which accelerate cellular ageing, impair DNA repair fidelity, and compromise host-pathogen defence. These stressors converge on gene expression and protein homeostasis in ways that are still poorly characterised in real microgravity. Conducting molecular biology experiments in space currently demands cold-chain infrastructure and complex equipment incompatible with deep-space payload constraints. A lightweight, room-temperature-stable biological laboratory would transform our ability to study and respond to these challenges in real time, on-orbit.

2. Molecular and Genetic Targets

Primary targets:

• RAD51 and BRCA2 — homologous recombination DNA repair genes; expression altered under ionising radiation and microgravity.
• NRF2 (NFE2L2) pathway transcripts — master regulator of oxidative stress response.
• Broad transcriptomic profiling via cell-free ribosome display and lateral flow readout as a low-mass omics proxy.

3. Target Relevance to the Space Biology Challenge

Radiation-induced double-strand breaks require RAD51-mediated homologous recombination for faithful repair; suppression of this pathway under microgravity increases mutation accumulation rates. NRF2 governs the antioxidant response to reactive oxygen species generated by cosmic radiation. Both pathways are dynamically regulated at the transcript and protein level, making them ideal targets for a cell-free expression-based sensing platform. Monitoring their activity in real time, using on-orbit synthesised reporters, would provide actionable data on crew molecular health without requiring live-cell culture or centrifuge-dependent assays.

4. Hypothesis and Research Goal

Hypothesis: A freeze-dried cell-free biosensor system, stabilised in trehalose matrix and embedded in mycelium-derived structural panels, can perform on-orbit transcriptomic monitoring of radiation-responsive and oxidative stress pathways (RAD51, NRF2) with sensitivity equivalent to bench-grade RT-qPCR, at a fraction of the mass and power budget.

Reasoning: CFPS reactions have been lyophilised and reactivated months later with retained fidelity. Mycelium composites provide structural, thermal, and radioprotective properties that passive aluminium panels cannot. Combining both technologies creates a laboratory architecture where the walls, benchtops, and insulation panels are themselves functional biological substrates, not passive enclosures. If validated, this platform collapses the payload mass requirement for a functional molecular biology laboratory by an order of magnitude.

5. Experimental Plan

Samples and model organisms

• Primary sample: Human saliva or fingerprick blood from crew members as minimally invasive nucleic acid sources.
• Biological model: Arabidopsis thaliana seedlings grown in mycelium substrate panels as a parallel plant stress model.
• Radioprotection model: Cladosporium sphaerospermum melanised fungal cultures integrated into habitat wall panels as living radioprotective layer.

Core experimental modules

Mycelium laboratory infrastructure

• Structural panels: Ganoderma lucidum mycelium grown on processed regolith simulant or cellulose waste; compression-moulded into benchtop, wall, and insulation panels.
• Radioprotective skin layer: Melanised Cladosporium sphaerospermum integrated into outer wall MBC composite; demonstrated on-orbit aboard the ISS to attenuate ionising radiation by up to 2.42-fold.
• Self-repair capacity: Living mycelium panels can re-colonise micro-fractures when rehydrated, reducing structural maintenance payload.
• Thermal insulation: MBC panels provide thermal insulation comparable to expanded polystyrene at one-third the density, critical for temperature-sensitive CFPS cartridge stability.

CFPS cartridge design

• Each cartridge is a replaceable unit containing lyophilised E. coli S30 extract, toehold switch plasmid, energy regeneration mix (PEP/pyruvate kinase), and amino acids.
• Activation: crew adds 15 to 30 microlitres of rehydration buffer (sterile water or saliva directly).
• Readout: fluorescence measured with a handheld LED torch and smartphone camera, or colorimetric readout read visually.
• Cartridge stability: 12 months at room temperature in sealed foil pouch; trehalose matrix validated for long-duration storage.
• Each cartridge is single-use, biodegradable, and compatible with mycelium composting for waste processing closure.

6. Addressing Space-Environment Constraints

7. Significance

MycoLab-1 addresses three converging needs in space exploration. First, it provides a credible molecular health monitoring platform for crew on multi-year missions beyond low Earth orbit where medical evacuation is not an option. Second, it demonstrates in-situ resource utilisation for laboratory infrastructure, growing structural and functional lab components from waste streams rather than Earth-launched payloads. Third, it creates a proof-of-concept for distributed biological laboratories in resource-constrained environments on Earth, including field hospitals, remote clinics, and low-income research institutions. The same system that monitors astronaut DNA repair fidelity on a Mars transit vehicle could monitor antibiotic resistance gene expression in a rural West African clinic.

Key Genes and Components Reference

Part B. Individual Final Project