Week 09 HW: cell free systems

1. Advantages of cell-free systems Cell-free protein synthesis (CFPS) offers a highly flexible and controllable environment compared to in vivo expression systems. Because there are no living cells, experimental conditions such as pH, ionic strength, redox environment, DNA concentration, cofactors, and additives can be directly tuned without affecting cell viability. This enables rapid optimization and prototyping of genetic constructs.

Additionally, CFPS is significantly faster, allowing protein production within hours instead of requiring cell growth, transformation, and induction steps.

Cell-free systems are particularly advantageous in cases such as:

• Toxic proteins: proteins that would inhibit or kill host cells can be produced safely
• Membrane proteins: can be expressed with detergents, liposomes, or nanodiscs to improve folding and functionality

2. Components of a cell-free system

A typical cell-free expression system includes:

Cell extract / TX-TL machinery

• Provides ribosomes, tRNAs, enzymes, and factors required for transcription and translation
• DNA or mRNA template - Encodes the protein of interest
• Amino acids Building blocks for protein synthesis
• Nucleotides (ATP, GTP, CTP, UTP) - Required for transcription and energy transfer
• Energy regeneration system - Maintains ATP/GTP supply during the reaction
• Buffer + cofactors (Mg²⁺, K⁺, etc.) - Maintain optimal biochemical conditions
• Optional additives (chaperones, lipids, detergents)- Help folding or membrane protein insertion

3. Why energy regeneration is critical

ATP and GTP are consumed during:

• transcription
• tRNA charging
• ribosomal translation
• Without regeneration, the reaction stops quickly.

Solution: Use an energy regeneration system such as: phosphoenolpyruvate (PEP) + pyruvate kinase or creatine phosphate + creatine kinase. These systems continuously regenerate ATP, allowing sustained protein production.

4. Prokaryotic vs eukaryotic systems

5. Designing a membrane protein experiment

Challenges:

• Poor solubility
• Misfolding
• Aggregation Approach:
• Add detergents or liposomes to mimic membranes
• Include chaperones
• Optimize Mg²⁺, temperature, and energy system

Homework question from Kate Adamala

Input: external signal (e.g. chemical or mechanical inducer) Output: cellulose-related components such as:

• cellulose synthase subunits
• UDP-glucose
• regulatory signals controlling cellulose production

b. Could this function be realized by cell-free Tx/Tl alone?

Partially, cell-free TXTL systems can produce proteins such as cellulose synthase subunits or regulatory molecules. However, full cellulose biosynthesis requires:

• membrane localization
• metabolic regeneration
• long-term energy supply

TXTL alone is insufficient for complete cellulose production, but suitable for prototyping and partial functionality.

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

Yes, and this is currently the most realistic approach. Organisms such as Komagataeibacter rhaeticus naturally produce bacterial cellulose and can be genetically engineered to control production using synthetic circuits (e.g. optogenetic systems). However, synthetic cells offer advantages in:

• controllability
• modularity
• reduced biological complexity

d. Desired outcome The goal is to create a programmable material production system where synthetic cells can spatially or temporally control cellulose formation, enabling structured biomaterials.

🧪 2. Design of the synthetic cell

a. Membrane The synthetic cell membrane would consist of:

• phospholipid bilayer vesicles (liposomes) e.g. POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)

b. Encapsulated components Inside the synthetic cell:

• TXTL system (e.g. E. coli extract)
• DNA encoding:
• cellulose synthase components (bcsA, bcsB)
• regulatory proteins
• nucleotides (ATP, GTP, etc.)
• amino acids
• energy regeneration system (e.g. PEP)
• cofactors (Mg²⁺, K⁺)

c. Source of TXTL system

A bacterial TXTL system (E. coli-based) is sufficient because:

• fast and high-yield
• compatible with most synthetic biology parts
• no need for eukaryotic post-translational modifications

d. Communication with environment The synthetic cell would interact with its environment through:

• passive diffusion (small molecules like glucose)
• embrane pores or channels, such as: α-hemolysin (forms pores in lipid membranes)

This allows uptake of substrates and release of products.

🧬 3. Experimental details a. Example components (genes + lipids) Lipids:

• POPC
• cholesterol Genes:
• bcsA (cellulose synthase catalytic subunit)
• bcsB (periplasmic subunit)
• optional regulators of cellulose synthesis
• α-hemolysin (for membrane permeability)

b. Measurement of function Function could be evaluated by:

• detecting protein expression (e.g. GFP fusion)
• measuring cellulose production using:
• Calcofluor staining
• dry weight measurement
• SEM microscopy to observe fiber formation

Homework question from Peter Nguyen

Based on my idea 1 for my final project I would develop a Bacterial cellulose cosmetic skinmask that would sense the “health” of the customers skin. Facemasks are populair single use product, however they are “dumb” providing a singulair batch of substances without telling you anything about what your skin acctually needs.

BC is already a compelling cosmetic substrate because it holds a lot of water, conforms well to skin, and has been tested as a moisturizing sheet mask material. In one evaluation, iinstead of putting living engineered cells on the face, a safer “synthetic biology” route is to embed freeze-dried cell-free gene expression (TX-TL) into the BC sheet as small patterned “sensor dots.” These cell-free circuits stay inactive when dry, then turn on when the mask hydrates during wear; outputs can be colorimetric (visible) or optical.

Because freeze-dried cell-free circuits activate upon rehydration, a conventional pre-hydrated sheet mask would trigger prematurely during storage. A practical design might be a dry-stored BC mask (or a separate paper sensor tab) that is activated only at time of use by releasing fluid.

How it could work:

• Input (skin/sweat biomarker): pH (skin barrier/irritation proxy), lactate (sweat/metabolic proxy).
• Sensing layer (cell-free circuit): a biomarker-responsive regulatory element controls whether a reporter is expressed.
• Output (visible color): express a chromoprotein (strong color under normal light) so the mask visibly shifts color in specific zones without any instrument; chromoproteins are attractive for “naked-eye” readouts.

The advantage of this concept is that facemask is already concidered as single use products so the one time use limitation of freeze dried system is becoming a desirable feature.

Homework question from Ally Huang

1. Background

My proposal is to develop a freeze-dried BioBits paper-based diagnostic for astronaut urine monitoring. The system would function as a “smart toilet paper” that rehydrates on contact with urine and produces a visible or fluorescent signal when a molecular marker of infection is present. This approach addresses the need for low-resource, non-invasive health monitoring in space, where medical infrastructure is limited. Urinary tract infections (UTIs) are a relevant risk due to immune changes in microgravity. This project is scientifically interesting because it combines synthetic biology, paper-based diagnostics, and cell-free systems for autonomous health monitoring.

3. Molecular / genetic target Bacterial 16S rRNA sequence specific to Escherichia coli as a biomarker for urinary tract infection.

4. Relation to space biology challenge Astronauts experience immune dysregulation and altered microbial behavior in microgravity, increasing susceptibility to infections. Urinary tract infections are particularly relevant due to hygiene constraints and closed environments during long-duration missions. Detecting bacterial 16S rRNA from Escherichia coli, a common UTI-causing organism, provides a direct molecular indicator of infection. A paper-based, cell-free diagnostic allows rapid, on-site detection without the need for complex laboratory equipment. This enables early intervention and reduces health risks, making it highly relevant for maintaining crew health during extended space travel.

5. Hypothesis / research goal I hypothesize that a freeze-dried BioBits cell-free system embedded in paper can detect bacterial RNA from Escherichia coli in urine and produce a measurable colorimetric or fluorescent output upon rehydration. The system would be designed with a DNA construct that responds to the presence of a target RNA sequence, triggering expression of a reporter protein such as GFP. The reasoning is that cell-free systems are stable when freeze-dried and can be activated by simple hydration, making them ideal for space applications. By integrating this system into a paper substrate, it becomes a lightweight, disposable diagnostic tool. The goal is to demonstrate that molecular detection and signal generation can occur reliably in a minimal, equipment-free format suitable for use in microgravity environments.

6. Experimental plan Urine samples spiked with Escherichia coli RNA will be applied to freeze-dried BioBits paper assays. Controls include: (1) urine without bacterial RNA (negative control) and (2) samples with known RNA concentration (positive control). The assay contains a DNA construct that produces a reporter signal in response to the target sequence. Upon rehydration, the reaction will be incubated and analyzed for color change or fluorescence using the P51 Molecular Fluorescence Viewer. Data collected will include signal intensity over time and detection sensitivity. This will assess the feasibility of rapid, paper-based molecular diagnostics in space.

PART B — Final Project Integration

Cell-free systems could be highly valuable for prototyping the optogenetic circuit before implementing it in Komagataeibacter rhaeticus. Instead of directly assembling and testing the full system in vivo (which is slow and complex), a cell-free system could be used to:

• Rapidly test Opto-T7RNAP activation dynamics
• Measure leakage in dark vs light conditions
• Optimize sRNA expression strength
• Tune arabinose induction levels
• Characterize response curves to projected light patterns

Because CFPS allows direct control over DNA concentration and reaction conditions, it would enable systematic testing of circuit parameters such as:

• promoter strength
• sRNA efficiency
• degradation rates
• transcriptional leakage

This would significantly reduce uncertainty before moving to in vivo experiments, where additional complexity (metabolism, diffusion, growth) makes debugging more difficult. In particular, cell-free systems could serve as a pre-validation layer for Aim 1, allowing partial validation of circuit logic even if full cellulose production cannot be reproduced in vitro.