Week 09: Cell-Free Systems

General Homework Questions

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

Cell-free protein synthesis (CFPS) eliminates the need to maintain viable cells, giving direct access to the reaction environment. You can tune pH, redox state, temperature, and add cofactors like chaperones or lipids directly — impossible inside a living cell.

Two Cases Where Cell-Free Beats In Vivo

Toxic proteins — antimicrobial peptides (e.g., magainin 2, human β-defensin-2) kill host bacteria. Cell-free has no host to protect.
Non-canonical amino acids (NCAAs) — NCAAs incorporated via amber codon suppression (TAG) and orthogonal tRNA synthetases do not need to cross a cell membrane, making CFPS the only practical route for site-specific unnatural amino acid incorporation.

2. Main Components of a Cell-Free Expression System

Component	Role
Cell extract (lysate)	Provides ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation/elongation factors
DNA template	Encodes protein of interest (plasmid or linear PCR product)
NTPs (ATP, GTP, CTP, UTP)	Powers transcription and translation
Amino acids	Building blocks for polypeptide synthesis
Energy regeneration system	Continuously regenerates ATP (e.g., PEP + pyruvate kinase)
Salts & buffer	Mg²⁺ and K⁺ maintain ribosome activity and optimal pH
T7 RNA polymerase (if needed)	Transcribes DNA → mRNA when not present in the lysate

3. Energy Regeneration in Cell-Free Systems

ATP and GTP are consumed rapidly during translation (one ATP per amino acid activation, two GTP per elongation cycle). Without regeneration, synthesis stops within 30–60 minutes.

Method: Phosphoenolpyruvate (PEP) / Pyruvate Kinase (PK) System

Pyruvate kinase transfers the high-energy phosphate from PEP to ADP:

PEP + ADP → ATP + pyruvate

Add 10–30 mM PEP + >10 U/mL pyruvate kinase to the reaction. This is the most widely used system in E. coli-based CFPS and provides >2× higher yield than creatine phosphate alone.

4. Prokaryotic vs. Eukaryotic Cell-Free Systems

	Prokaryotic (E. coli)	Eukaryotic (wheat germ / rabbit reticulocyte)
Cost	Low	High
Yield	High (mg/mL)	Moderate
PTMs	None	Glycosylation, disulfide isomerization
Speed	Fast (2–4 h)	Slower
Best for	Bacterial proteins, rapid screening	Mammalian glycoproteins, complex folds

Examples

Prokaryotic choice: Renilla luciferase — no PTMs needed, folds well in E. coli extract, widely used as a reporter in CFPS optimization.
Eukaryotic choice: Human erythropoietin (EPO) — requires N-linked glycosylation for stability and activity; only eukaryotic lysates can add these glycans correctly.

5. Optimizing Membrane Protein Expression in Cell-Free Systems

The challenge with membrane proteins in CFPS is that they are hydrophobic and aggregate in aqueous reaction mixtures. The solution is to add lipid scaffolds directly into the reaction.

Setup

Nanodiscs — Pre-assemble MSP1D1 + DMPC or POPC nanodiscs and add them directly to the CFPS reaction. Nascent membrane proteins insert co-translationally into the disc. Nanodiscs outperform detergents and liposomes for solubility.
Reduce translation rate — Use lower DNA concentration (1–5 nM) or a weaker RBS to slow elongation, giving transmembrane helices time to insert.
Detergents as alternatives — DDM or digitonin can be added above their CMC to solubilize the protein, but screen carefully because many detergents inhibit CFPS at higher concentrations.
Measurement — GFP-fusion + size-exclusion chromatography to confirm monodisperse, folded protein.

6. Troubleshooting Low Yield in Cell-Free Systems

Cause	Diagnosis	Fix
Template degradation	Run gel of lysate + plasmid after 1 h; check for smearing	Add RNasin (RNase inhibitor); use nuclease-deficient extract strain
Energy depletion	Time-course shows synthesis stops before 1 h	Increase PEP concentration; switch to maltose-based system
mRNA secondary structure	Mfold/RNAfold predicts strong 5′ hairpin	Introduce synonymous mutations; test alternate 5′ UTRs

Kate Adamala — Synthetic Minimal Cell Design

Concept: Biofilm-Sensing, Quorum-Quenching Synthetic Cell

1a. Function — Input and Output

Input: N-Acyl homoserine lactones (AHLs) secreted by biofilm-forming pathogens such as Pseudomonas aeruginosa
Output: Release of AHL lactonase (AiiA, encoded by aiiA) to degrade quorum-sensing signals and halt biofilm maturation

1b. Cell-Free Tx/Tl Alone Without Encapsulation?

No. Without encapsulation, lactonase diffuses freely into the environment with no threshold-gated release. Encapsulation couples AHL sensing to controlled output release, giving logical behavior.

1c. Could a Genetically Modified Natural Cell Do This?

Yes — E. coli can be engineered with a LuxR-responsive aiiA circuit. However, natural cells carry risks of uncontrolled replication, immune activation, and off-target effects. Synthetic cells are non-replicating and immunologically inert.

1d. Desired Outcome

Synthetic cells sense AHL above threshold → express α-hemolysin pores → release AiiA lactonase → degrade AHL → disrupt quorum sensing → biofilm bacteria become antibiotic-sensitive.

2a. Membrane Composition

POPC + cholesterol (70:30 ratio)

2b. Encapsulated Contents

PURE system (bacterial transcription/translation)
Gene: aiiA (AHL lactonase from Bacillus sp. 240B1)
Gene: hla (α-hemolysin from Staphylococcus aureus)
LuxR protein (constitutively present)

2c. Transcription/Translation System

Bacterial PURE system

AHL-LuxR signaling is bacterial, so a bacterial Tx/Tl system is the correct choice.

2d. Communication With Environment

AHL molecules passively diffuse across the membrane. Once activated, α-hemolysin forms pores in the membrane, allowing AiiA lactonase to exit and degrade extracellular AHL.

3a. Genes and Lipids

Lipids

POPC
Cholesterol

Gene 1 — aiiA

AHL lactonase from Bacillus sp. 240B1
GenBank: AF196151
Function: hydrolyzes the lactone ring of AHL molecules

Gene 2 — hla

α-hemolysin from Staphylococcus aureus
UniProt: P09616
Function: forms heptameric membrane pores

Regulator — luxR

LuxR transcriptional activator from Vibrio fischeri
UniProt: P12746
Activated by AHL and drives the lux promoter

3b. Measurement

Crystal Violet Biofilm Assay

Treat Pseudomonas aeruginosa biofilms with synthetic cells and compare against a no-AHL control.

Measure OD570 after crystal violet staining
Reduced staining indicates biofilm degradation

Secondary Assay

SYTO9/PI fluorescence microscopy to confirm bacterial sensitization and membrane integrity changes.

Peter Nguyen — Cell-Free Systems in Materials

Application: Smart Architectural Wall Panel

Pitch

A freeze-dried cell-free biosensor panel embedded into interior wall tiles changes color when indoor formaldehyde exceeds safe limits (>0.1 ppm, WHO threshold).

Mechanism

The tile contains lyophilized BioBits® pellets encoding a frmR-regulated colorimetric circuit.

Without formaldehyde → FrmR represses reporter expression
With formaldehyde → repression removed → β-galactosidase expressed
CPRG substrate changes color from yellow → blue

The user sprays water + CPRG to activate the tile. Color becomes visible within 2–4 hours.

Societal Need

Formaldehyde from furniture, flooring, and paint is a major indoor air pollutant linked to respiratory disease and cancer. Current electronic monitors cost >$100. This biosensor provides an inexpensive visual alternative.

Addressing Limitations

Stability: Lyophilized with trehalose + BSA for room-temperature storage
One-time use: Replaceable modular sensor inserts
Water activation: Prevents accidental activation from ambient humidity

Ally Huang — Mock Genes in Space Proposal

Background

Microgravity causes rapid skeletal muscle atrophy in astronauts. Pax7, a master regulator of satellite cell activation, becomes downregulated during spaceflight. Monitoring Pax7 expression in real time could help track muscle health and optimize countermeasures. Existing approaches require laboratory infrastructure unsuitable for space missions. A freeze-dried, field-deployable biosensor for Pax7 mRNA would enable portable muscle-health monitoring without refrigeration or complex equipment.

Molecular Target

Pax7 mRNA detected using a toehold switch reporter within a BioBits® cell-free reaction.

Target-Challenge Relationship

Pax7 expression is a direct indicator of muscle satellite cell activation and regenerative capacity. Microgravity suppresses mechanical loading, reducing Pax7-positive satellite cells. Measuring Pax7 mRNA abundance enables quantitative tracking of muscle regeneration status during long-duration missions.

Hypothesis

A freeze-dried BioBits® toehold switch biosensor targeting Pax7 mRNA will generate GFP fluorescence proportional to Pax7 expression levels in astronaut RNA samples, detectable using the P51 Molecular Fluorescence Viewer.

Toehold switches are programmable RNA sensors capable of recognizing nearly any target sequence. A Pax7-specific switch is incorporated into the BioBits® system so GFP translation occurs only in the presence of Pax7 transcript.

The miniPCR® device amplifies and transcribes RNA into a compatible format for the switch. This keeps the workflow entirely within the Genes in Space toolkit.

Low Pax7 → weak GFP signal
High Pax7 → strong GFP signal

Experimental Plan

Samples

Saliva or biopsy RNA from astronauts at:
- T = 0 (preflight)
- T = 30 days
- T = 60 days
- T = 90 days

Controls

Positive: synthetic Pax7 mRNA spike-in
Negative: nuclease-free water
Blank: osmolality-matched control

Procedure

Extract RNA using compact lysis kit
Use miniPCR® for RT-PCR amplification
Add amplicon to rehydrated BioBits® toehold pellet
Incubate 2 h at 37°C
Visualize fluorescence using P51 Viewer