CFPS Systems

General Questions

Reference paper: Cell-Free Gene Expression: Methods and Applications

1. Main advantages of cell-free protein synthesis (CFPS) systems over traditional in vivo methods

According to the course material, CFPS systems offers more flexibility because there is no need anymore to maintain cells alive. For instance, it allows the production of toxic proteins that would otherwise kill the cells producing them. Using easier cell machinery systems can also give scientists more flexibility in adapting their experimental design. For instance, it allows them to incorporate non-natural amino acids into the reaction. And finally, CFPS systems also offer much easier storage conditions because it is possible to freeze-dry the reactions on tiny pellets.

Cell-free protein synthesis also offers more control during production: there is no unknown cellular process occurring in parallel, nor interference with other proteins naturally produced by the cells. The conditions of the environment (e.g. ions concentrations, pH, enzymes etc.) can be defined more precisely. Finally, it makes it much easier to isolate the target protein. These assets are useful in the pharmaceutical industry for producing large batches of hormones (e.g. insulin), vaccines or medicines.

To go further…

A critical comparison of cellular and cell-free bioproduction systems

Cell-Free Synthesis: Expediting Biomanufacturing of Chemical and Biological Molecules

2. Main components of a CFPS system

Components of a CFPS reaction mixed in a test tube:

From Cell-free protein synthesis and vesicle systems for programmable therapeutic manufacturing and delivery

The CFPS system mixture contains:

• DNA template (linear or plasmid): gene of the protein of interest
• Cell extract that contains RNA polymerase, enzyme that synthesizes RNA from DNA; ribosomes, molecular machines that reads the mRNA and assembles the amino acids into a protein, transfer RNAs, adapter molecules that carry amino acids and match them to the corresponding codons on the mRNA during translation and further enzymes needed for the protein synthesis.
• Amino acids: building blocks of the proteins
• Free nucleotides: building blocks of the mRNA (A,C,G, U)
• Co-factors: maintain enzyme activity and ensure proper protein folding.
• Energy source: ATP, GTP and their regenerating substrates (e.g. PEP) provide the energy required for both transcription and translation processes.
• Salts: provide the ions needed for optimal enzyme activity and structural stability of the system.

3. Energy regeneration in cell-free systems

The translation and transcription processes require a lot of energy. Without a continuous supply in ATP, the protein synthesis quickly shuts down. Thus, energy regeneration is needed to provide a continuous supply of energy and avoid the accumulation of by-products (e.g. phosphate) that can interfere with the protein synthesis.

There are many different ways to regenerate energy in cell-free systems: see references. Choosing glucose as an energy source can be interesting in the prototyping phase of a project because it is a highly cost-effective and an efficient way to ensure a continuous supply of ATP via glycolysis in both E. coli and yeast extracts. It can also be combine with creatine phosphate in a dual system: creatine phosphate acts as a high-energy phosphate donor for rapid ATP regeneration via creatine kinase, while glucose feeds the glycolytic pathway to produce ATP and consume inorganic phosphate.

From The cost-efficiency realization in the Escherichia coli-based cell-free protein synthesis systems

References

ATP Regeneration from Pyruvate in the PURE System

Cell-Free PURE System: Evolution and Achievements

Development of prokaryotic cell-free systems for synthetic biology (includes PANOx energy regeneration system)

4. Prokaryotic vs. eukaryotic cell-free expression systems

Prokaryotic cell-free systems are fast and efficient for producing simple proteins. For instance, E.coli systems are ideal for producing simple proteins such as Green Fluorescence Protein (GFP) in labs and in the industry.

On the other hand, eukaryotic systems are slower and more expensive, but they can be used for the production of proteins which require complex folding, disulfides bridges, and post-translational modifications (e.g. glycosylation, lipidation) such human anti-bodies.

Reference

Protein Synthesis in Prokaryotes vs. Eukaryotes: What’s the Difference?

Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

5. Cell-free systems for the synthesis of membrane proteins

Membrane proteins are composed of transmembrane domains which makes them hydrophobic and thus hard to express in traditional cell-based systems: they need a special environment to avoid aggregation, and enable the proteins to fold and function correctly. However, it is possible to recreate such membrane-like environments by adding specific supplements to the reaction depending on the protein (Is it small and simple, or bigger and complex?) and what we want to do with it (do we only want to extract the protein or does it have to be functional?).

Cell-free systems derived from prokaryotic, as well as eukaryotic extracts that lack endogenous microsomes (natural membrane fragments from the cells) be can be supplemented with:

• Detergents, commonly used for solubilization and fast extraction membrane proteins.
• Nanodiscs, small membrane-like structures of adaptable size and easy to purify. Useful for stabilizing and studying the structure of the proteins.
• Liposomes, tiny vesicles composed of bilayer membranes that are more difficult to purify but also more adapted for transporter proteins and the ones that need to be oriented in the membrane. Useful for testing the function of the proteins.

From Cell-free Membrane Protein Synthesis

For my final project “Sensing perimenopause: a bioluminescent art installation”, one design strategy is to use the G protein–coupled receptor to elicit a bioluminescent response to an environmental change, e.g. a change in extracellular levels of glycogen in the vaginal secretions. If using a cell-free system, I might want to use eukaryotic cell extracts and add liposomes to ensure that the receptor can sense the glycogen levels in real-time.

To go further…

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

Membrane protein production in Escherichia coli cell-free lysates

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

6. Troubleshooting Low Protein Yield

Achieving optimal protein yield is a major challenge in cell-free protein synthesis systems. Below is a table that lists three common issues that can lead to low protein production and how to solve them.

References

How to Troubleshoot Low Protein Yield After Elution

Solved: Low Yields in Cell-Free Protein Synthesis

Troubleshooting Protein Folding Issues in Cell-Free Synthesis: Tips from Industry Experts

Synthetic Cell Design

1. Oestradiol Biosensor with Bioluminescent Output

1a. Biosensor Description

I would like to design a synthetic cell that can continuously monitor the extracellular concentration in oestradiol such (e.g. 17 β-oestradiol) and emit a quantifiable bioluminescent signal whose intensity is proportional to the oestradiol concentration.

Input: oestradiol concentration

Output: bioluminescent signal

1b. Cell-Free System vs Encapsulation

The design may function in a cell-free system, but encapsulation would probably improve:

• The stability and overall duration of the experimental conditions, usually limited to 2-6 hours in cell-free systems
• The signal-to-noise ratio by inducing a stronger output signal

1c. Oestradiol Biosensor in Genetically Modified Natural Cell

The oestrogen biosensing function has already been realized in genetically modified natural cells: estradiol-inducible gene expression systems (see GEV example below) have been created in yeast.

GEV: special hybrid protein that can switch on the expression of selected genes in yeast when binding to estradiol. GEV is made of three parts: a GAL4 DNA-binding domain (from yeast, can attach to specific portions of the DNA), Human estrogen receptor domain (detects β-estradiol) and VP16 activation domain (from herpes virus, activates gene expression). References: Louvion et al. (1993) Ottoz et al. (20214)

1d. Desired Outcome of the Synthetic Cell Operation

Upon exposure to oestradiol, the synthetic cells produce a sustained, concentration-proportional bioluminescent signal via e.g. NanoLuc luciferase (NLuc) acting on its substrate furimazine.

NanoLuc offers >150-fold increase in luminescence compared to established luciferase systems, along with enhanced stability and a smaller size (19 kDa). Reference: NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence

2. Components

Image credit: Kate Adamala’s lab

2a. Membrane Composition

According to Kate Adamala’s lecture, the membrane of the synthetic cell should be made of phospholipids and cholesterol.

2b. Encapsulated Contents

• Cell-free Tx/Tl system (e.g. E. coli PURE system) incl. RNA polymerase, co-factors, ribosomes, tRNAs, amino acids, ATP/GTP regeneration system
• Plasmid DNA encoding the genetic circuit (e.g. E2 sensing > expression of NLuc, see above)
• Pre-synthesised transcription factor protein (e.g. GEV) to accelerate the sensing response
• Possibly NLuc’s substrate (e.g. furimazine) acting as small reservoir before the intra- and extra-cellular concentrations equilibrate naturally

2c. System Type

Normally, human hormone receptors need many helper proteins and complicated processes inside human cells to work properly. But when integrating the GEV system, the protein can directly recognize and bind to the hormone, so there is no need for complicated cell machinery.

2d. Environmental Communication

• Extracellular oestradiol (input molecule) passively diffuses across the lipid bilayer due to its high lipophilicity, so there is no active transporter required here.
• The membrane is also permeable to furizamine (NLuc substrate).
• While the membrane is not an obstacle to the bioluminescent emission (photons), one needs to consider how the composition of the extracellular environment may affect the intensity of the output signal.
• One may consider adding nanopores to the membrane to enhance signal if oestradiol/furizamine might become limiting factors, but this would have to be finely tuned as nanopores could also cause components of the cell-free system to leak out.

3. Experimental Details

3a. Lipids and Genes

Giant Unilamellar Vesicles (GUVs) will be produced by emulsion phase transfer or microfluidic double-emulsion encapsulating the E. coli PURE system + DNA + pre-made GEV protein + furimazine.

Lipids:

According to Claude:

Genes:

• Gene 1 : Chimeric transcription factor (sensor module): GEV, Z₃EV or LexA-HBD(hERα)-B42). This protein is pre-made and encapsulated (part of GUV formation) to bypass the lag of de novo expression.
• Gene 2 : Reporter (output module): NanoLuc under the control of a synthetic promoter containing LexA operator arrays.
• Gene 3 : Repressor module: LexA-HBD(hERα)-KRAB (to implement a BAND-PASS circuit: at high oestradiol concentrations, the activator becomes out-competed by the repressor, suppressing NLuc expression above a saturation threshold).
• Gene 4 (optional) : Nanopores: hla (low expression, for furimazine equilibration without lysis).

3b. Output Measurements

The bioluminescent signal is detected by a luminometer, bioluminescence imaging system, or photonic sensor.

Chart created with ChatGPT. Reference: Instrumentation for Chemiluminescence and Bioluminescence

Measurement protocol suggested by Claude:

• Instrument calibration
• Single-vesicle imaging: Use bioluminescence microscopy to visualise individual GUVs responding to E2 gradients. This validates encapsulation efficiency and cell-to-cell signal heterogeneity.
• Selectivity control: Test against structurally related steroids (e.g. testosterone, progesterone, cortisol, oestrone E1, oestriol E3) at equimolar concentrations to confirm specificity of the binding for E2.
• Band-pass validation: Confirm that signal returns to baseline above a certain threshold.
• Negative controls: for instance, absence of plasmid, denatured GUV.

Freeze-Dried CFS

1. Bioluminescent TSS-Toxin Biosensor Tampons

Toxic shock syndrome (TSS) is a rare, life-threatening, toxin-mediated infectious process linked, in the vast majority of cases, to toxin-producing strains of Staphylococcus aureus or Streptococcus pyogenes. The project aim is to integrate a freeze-dried cell-free biosensor system into mycelium-based menstrual products to produce real-time bioluminescent signals upon detecting TSS-toxin-producing bacterial metabolites, enabling users to identify dangerous pathogenic activity before clinical symptoms emerge.

2. Mechanism

The tampon is made from biocompatible mycelium that forms a soft, porous, and absorbent material similar to the coton material used in conventional tampons. A cell-free biological system designed to detect the early signs of infection is embedded within this structure. The material contains the molecular components needed for sensing, including ribosomes, amino acids, and synthetic DNA instructions that activate the detection process when exposed to menstrual fluid. See Synthetic Cell Design HW section for details about the molecular biosensing principle.

The biosensor is designed to detect two harmful bacterial toxins: (1) Staphylococcus aureus toxic shock syndrome toxin-1 (TSST-1) and Streptococcus pyogenes streptococcal pyrogenic exotoxins (SPEs). It can do this either directly, by responding to bacterial toxins, or indirectly, by sensing bacterial byproducts that build up in the menstrual environment. When the tampon is inserted, menstrual fluid naturally rehydrates the freeze-dried biological components inside the material. This activates the monitoring of the vaginal environment and the detection of potential signs of infection.

3. Societal challenge

TSS remains a serious and often overlooked health risk. Although it is relatively rare in developed countries, it still carries a mortality rate of around 5–15%, and some survivors experience long-term damage to multiple organs (incl. amputation). One of the biggest challenges is that the early symptoms are vague and can look similar to the flu, making TSS difficult to recognise quickly. By the time it is diagnosed, severe complications may already have developed. The critical opportunity for intervention is within the 12–48 hour period after toxin exposure, before widespread inflammation and organ failure occur, but this window is often missed by both users and healthcare providers.

Current gap: There is currently no simple, accessible early warning system available. The existing vaginal health apps can only track symptoms after they appear and cannot detect harmful bacteria or toxins in real time. Biomarker testing is not currently available for home use. The diagnosis still depends on laboratory cultures, which can take 3–5 days for results.

The project can have a positive clinical impact. Detecting sepsis even 12 hours earlier can make a major difference: earlier diagnosis improves patient outcomes and helps reduce healthcare costs.

It can also improve health equity. The tampon-based warning system offers a simple, non-invasive way for menstruating people to detect early signs of infection. It could be especially helpful in low-resource areas where access to hospitals or medical care may be delayed.

Finally, the project seems to be a good fit with the current market needs. Indeed, the success of personal health products such as glucose monitors, ovulation trackers and pregnancy tests shows that people are comfortable using health diagnostics in private settings.

Sources: Claude + Toxic Shock Syndrome: A Literature Review. Antibiotics (2024) https://pubmed.ncbi.nlm.nih.gov/38247655

4. Cell-free system limitations

Table created with Claude.

Freeze-Dried CFS in Space

Glow for Life: Detecting Biosignatures in Microgravity

Background

Long-duration space missions require compact systems capable of detecting possible biosignatures in extraterrestrial environments. Freeze-dried cell-free protein synthesis (CFPS) systems such as BioBits® are promising because they remain stable without refrigeration and function in microgravity. Rather than searching for specific organisms, this project investigates whether cell-free biosensors can detect molecular patterns associated with life-like chemistry, including nucleic-acid-like sequences and ATP-dependent enzymatic activity. Developing lightweight biosignature detection systems is important for future missions to Mars, icy moons, and returned planetary samples, while also improving portable diagnostic technologies for remote environments on Earth.

Molecular or Genetic Target

ATP-dependent fluorescence activation and synthetic RNA trigger sequences detectable by BioBits® cell-free transcription–translation reactions.

Relationship of the Target to the Challenge

ATP is used by all known life on Earth as a molecule for energy transfer, making ATP-related biochemical activity a strong candidate for a general biosignature. Synthetic RNA trigger sequences can also be used to test whether BioBits® biosensors maintain their sensitivity and specificity in microgravity conditions. If these cell-free systems can reliably detect biologically relevant molecules in space, they could become portable screening tools for future astrobiology missions. This experiment does not assume that extraterrestrial life would use the same genetics as life on Earth. Instead, it investigates whether stable molecular indicators associated with metabolism or information-carrying polymers can be detected using lightweight, freeze-dried biosensors that are compatible with spacecraft limitations in mass, power, and containment.

Hypothesis / Research Goal

This project tests the hypothesis that freeze-dried cell-free biosensors retain sufficient sensitivity and specificity in microgravity to detect molecular signatures associated with life-like biochemical activity. Previous ISS experiments demonstrated that BioBits® can successfully express fluorescent proteins and RNA biosensors in orbit, confirming that cell-free transcription and translation remain functional under spaceflight conditions.

The research goal is to evaluate whether these systems can be adapted into generalized biosignature detectors suitable for future planetary exploration missions. Fluorescent outputs generated after exposure to ATP-containing samples or synthetic RNA targets would indicate successful biosensor activation. Negative controls lacking target molecules should show minimal fluorescence. Demonstrating reliable operation of freeze-dried biosensors in space would support future development of compact astrobiology instruments for missions where mass, power, and biological containment are limited.

Experimental Plan

Freeze-dried BioBits® reactions containing fluorescent reporter constructs will be rehydrated with: (1) ATP-positive samples, (2) synthetic RNA trigger samples, and (3) negative-control samples lacking targets. Additional controls will include degraded ATP and randomized RNA sequences to test specificity. Reactions will be incubated aboard the ISS and fluorescence measured using the P51™ Molecular Fluorescence Viewer. Optional miniPCR® amplification of synthetic RNA targets can verify sequence-dependent activation. Quantitative fluorescence intensity and reaction timing will be compared between flight and ground controls to determine whether microgravity alters biosensor sensitivity, specificity, or reaction kinetics.