Week 9 Week 9 — Cell-Free Systems

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.

Advantages of cell-free protein synthesis (CFPS) over traditional in-vivo methods:

(i) Greater flexibility and control: Given that cells do not need to stay “alive” and the absence of a cell wall, it is possible to manipulate cells in real time; add chaperones, cofactors etc [1].

(ii) Rapid development of prototypes: Where in-vivo methods require cloning DNA into plasmids and transforming host cells, the CFPS allows us to essentially ‘drag and drop’ DNA with raw PCR products and observe protein expression in short periods of time (e.g. hours) [2]

Cases where CFPS provides benefits over in-vivo methods:

(i) Expression of toxic/dangerous antimicrobial peptides, potent neurotoxins, or complex membrane proteins in vivo. Usually the host cell would ‘die’ before reaching a large protein yield, as the CFPS is technically dead, it can synthesize toxic therapeutics and viral vectors that would be impossible to harvest from living cultures [2]

(ii) The open environment lets you easily swap natural amino acids for synthetic ones, enabling efficient, site-specific incorporation of non-standard amino acids (nsAAs) without competing with host metabolism [2]

  1. Khambhati K, Bhattacharjee G, Gohil N, Braddick D, Kulkarni V, Singh V. Exploring the potential of cell-free protein synthesis for extending the abilities of biological systems. Front Bioeng Biotechnol. 2019;7:248.

  2. Silverman AD, Kelley-Loughnane N, Jewett MC. Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet. 2020;21(3):151-70.

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

(i) Cell extract (machinery): Derived from lysed cells (like E. coli), this extract provides the core transcriptional and translational machinery, including ribosomes and RNA polymerase, required to build the protein

(ii) Genetic template (blueprint): The DNA plasmid or RNA template that contains the specific gene sequence of the target protein we want to express

(iii) Nucleotides and amino acids (building blocks): Nucleotides—Adenosine triphosphate (ATP), Guanosine triphosphate (GTP), Cytidine triphosphate (CTP), and Uridine triphosphate (UTP)—are supplied for ribonucleic acid (RNA) synthesis (transcription), while transfer RNAs (tRNAs) pair with messenger RNA (mRNA) to deliver the amino acids necessary for protein synthesis (translation)

(iv) Energy systems: immediate energy sources like adenosine triphosphate (ATP) are paired with intermediate metabolites like 3-phosphoglycerate (3-PGA) or phosphoenolpyruvate (PEP) to continuously regenerate energy and maintain reaction stability.

(v) Buffers & cofactors (Environmental conditions):

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (buffer to maintain a stable pH for optimal enzyme activity)

Mg: Magnesium (cofactor for transcription and translation enzymes)

DTT: Dithiothreitol (reducing agent that maintains a non-oxidizing environment to protect protein residues)

Sodium Oxalate: This is already the full chemical name (there is no abbreviation here, though its chemical formula is Na₂C₂O₄) (prevent magnesium precipitation, stabilizing the ionic balance)

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

Transcription and translation consume ATP/GTP rapidly, and the by-product inorganic phosphate chelates Mg²⁺, stalling ribosomes within roughly 1 hour without replenishment.

For continuous ATP supply, a possible system such as phosphoenolpyruvate (PEP) + pyruvate kinase, or creatine phosphate + creatine kinase, continuously re-phosphorylates ADP to ATP, sustaining synthesis for several hours.

Filippo Caschera, Vincent Noireaux. Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie. 2014 Apr;99:162-168. doi: 10.1016/j.biochi.2013.11.025. Epub 2013 Dec 8. PMID: 24326247

4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. Prokaryotic systems such as E. coli S30 give high yields, are low cost, and have fast turnaround but lack post-translational modifications (PTMs); eukaryotic systems (wheat germ, CHO, HeLa) yield less but support glycosylation, disulfide bonds, and complex folding.

Therefore choosing:

GFP as it folds autonomously, no PTMs, ideal for rapid high-yield prototyping.

Eukaryotic choice: erythropoietin (EPO) as it requires N-glycosylation and disulfide bonds for activity, only achievable in a mammalian lysate

Anne Zemella, Lena Thoring, Christian Hoffmeister, Stefan Kubick. Cell-free protein synthesis: Pros and cons of prokaryotic and eukaryotic systems. ChemBioChem. 2015 Nov;16(17):2420-2431. doi: 10.1002/cbic.201500340. Epub 2015 Oct 19. PMID: 26478227; PMCID: PMC4676933.

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:

(i) Hydrophobic domains aggregate in aqueous mixes, the protein needs a lipid-like environment to fold

(ii) This could be toxic in-vivo

Design:

(i) Template: T7-driven, His-tagged construct of the membrane protein

(ii) Extract: E. coli S30 lysate

(iii) Supplement (test in parallel): mild detergents (Brij-35, DDM), nanodiscs (MSP + lipids), or liposomes

(iv) Optimise: Mg²⁺, K⁺, and temperature in a small factorial screen

Validation:

(i) SDS-PAGE + anti-His Western blot to confirm expression

(ii) Ultracentrifugation to separate soluble vs membrane-inserted fractions

(iii) Functional or ligand-binding assay to confirm native folding

Daniel Schwarz, Friederike Junge, Florian Durst, Nadine Frölich, Birgit Schneider, Sina Reckel, Solmaz Sobhanifar, Volker Dötsch, Frank Bernhard. Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems. Nat Protoc. 2007;2(11):2945-2957.

  1. 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.

(i) Energy depletion: add a regeneration system (PEP/pyruvate kinase or creatine phosphate/creatine kinase) or use a continuous-exchange (CECF) reactor.

(ii) Misfolding or proteolysis: lower temperature to 25 °C, add chaperones (GroEL/ES, DnaK) and protease/RNase inhibitors

(iii) Inefficient setup: re-purify DNA, check integrity on gel, ensure a T7 promoter and strong RBS, titrate 5–20 ng/µL

Adam D. Silverman, Ashty S. Karim, Michael C. Jewett. Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet. 2020 Mar;21(3):151-170. doi: 10.1038/s41576-019-0186-3. Epub 2019 Nov 28. PMID: 31780816.

Homework question from Kate Adamala

Function

A synthetic minimal cell that expands gut-brain axis signalling.

Input: Tumor Necrosis Factor-alpha (TNF-α), elevated in intestinal inflammation.

Output of SMC: 5-hydroxytryptophan (5-HTP), a serotonin precursor. Output of whole system: increased serotonin production in enterochromaffin cells, improving mood-relevant signalling.

Could this be cell-free transcription/translation (Tx/Tl) without encapsulation?

No, encapsulation is required to spatially contain the enzymatic conversion of tryptophan to 5-HTP, preventing uncontrolled release and ensuring TNF-α-triggered production only.

Could a genetically modified natural cell do this?

Yes, but SMCs offer safer, non-replicating, controllable delivery without risk of horizontal gene transfer or colonisation.

Desired outcome: In the presence of intestinal inflammation, SMCs locally produce and release 5-HTP, dampening the inflammation-serotonin deficit link in the gut-brain axis.

Components

Membrane:

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + cholesterol

Encapsulated:

Bacterial cell-free Tx/Tl; tryptophan hydroxylase 1 gene (TPH1) under TNF-α-responsive promoter; tryptophan (substrate)

Tx/Tl system:

Bacterial (TNF-α responsive elements achievable with engineered promoters)

Communication:

TNF-α diffuses into SMC; 5-HTP exits via alpha-hemolysin (aHL) pore expressed upon TNF-α sensing

Experimental details

Lipids: POPC, cholesterol

Genes: TPH1 (tryptophan hydroxylase 1); aHL (alpha-hemolysin membrane pore)

Small molecules: tryptophan (encapsulated substrate)

Measurement:

Enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography (HPLC) for 5-HTP output; serotonin levels in co-cultured enterochromaffin cells

  1. Paul Strandwitz. Neurotransmitter modulation by the gut microbiota. Brain Res. 2018;1693(Pt B):128-133. doi:10.1016/j.brainres.2018.03.015.

  2. Júlia Leão Batista Simões, Geórgia de Carvalho Braga, Charles Elias Assmann, Margarete Dulce Bagatini. Targeting the gut-immune-brain axis: pharmacological insights from depression in inflammatory bowel disease. Front Pharmacol. 2026 Apr 1;17:1793292. doi:10.3389/fphar.2026.1793292. PMID: 41993582; PMCID: PMC13079007.

Homework question from Peter Nguyen

Freeze-dried cell-free systems can be incorporated into all kinds of materials as biological sensors or as inducible enzymes to modify the material itself or the surrounding environment. Choose one application field — Architecture, Textiles/Fashion, or Robotics — and propose an application using cell-free systems that are functionally integrated into the material. Answer each of these key questions for your proposal pitch:

• Write a one-sentence summary pitch sentence describing your concept.

• How will the idea work, in more detail? Write 3-4 sentences or more.

• What societal challenge or market need will this address?

• How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

Walls or tiles in homes in arsenic-endemic regions embedded with cell-free biosensors that visibly indicate arsenic contamination that change colour when water containing arsenic flows over or is applied to them.

• Freeze-dried cell-free systems containing ArsR biosensor embedded into a porous tile or panel surface coating

• HH member applies/splashes water sample onto the tile

• Water rehydrates the cell-free system

• If arsenic present –> colour change visible to the naked eye | can be a “testing wall”

Works towards one contributor of chronic kidney disease from arsenic. Prevalence is higher among communities dependent on communal wells. Additionally, no behaviour change would be needed, it would be part of regular chores/tasks.

Addressing cell-free system limitations: Activation with water: Naturally solved through water sample application is the intended use, making rehydration a feature rather than a limitation Stability: Freeze-drying into a protective hydrogel matrix embedded within the ceramic tile pores confers long shelf-life; tiles can be stored and installed in hot climates without refrigeration, as freeze-dried cell-free systems have demonstrated stability at ambient temperatures for extended periods

Homework question from Ally Huang

Freeze-dried cell-free reactions have great potential in space, where resources are constrained. As described in my talk, the Genes in Space competition challenges students to consider how biotechnology, including cell-free reactions, can be used to solve biological problems encountered in space. While the competition is limited to only high school students, your assignment will be to develop your own mock Genes in Space proposal to practice thinking about biotech applications in space! For this particular assignment, your proposal is required to incorporate the BioBits® cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCR® thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/.

(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. (Maximum 100 words)

Future missions to Mars and icy moons such as Europa will be unable to return contaminated samples to Earth for analysis, requiring an on-site biological screening tool. A rapid, equipment-minimal, on-site biological screening tool for astronaut safety and planetary protection which would be in the form of freeze-dried cell-free systems offer a uniquely stable, rehydration-activated solution deployable without refrigeration or living cells across multi-year deep space missions.

(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. (Maximum 30 words)

Pathogen-specific messenger RNA (mRNA) sequences from Pseudomonas aeruginosa and Salmonella, detected via toehold switch riboregulators.

(3) Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)

Toehold switches are synthetic riboregulators that only trigger translation of a fluorescent reporter when a specific target RNA is present. Embedding these into the BioBits cell-free system creates a programmable, rehydration-activated biosensor that can be reprogrammed for different pathogens or extraterrestrial biosignatures.

(4) Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)

Freeze-dried BioBits cell-free systems incorporating pathogen-specific toehold switches can be rehydrated with extraterrestrial liquid samples and produce a detectable fluorescent signal within 2–3 hours, enabling rapid on-site pathogen screening without living cells or complex equipment, even under microgravity conditions.

(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. (Maximum 100 words)

(i) Rehydrate toehold switch BioBits reactions with pathogen RNA spiked into simulated Martian brine and Europa ocean analogue solutions

(ii) Pre-amplify trace nucleic acids using miniPCR where sample concentrations are too low for direct detection

(iii) Visualise and quantify fluorescent output using the P51 Molecular Fluorescence Viewer

(iv) Controls: sterile water (negative) and non-target RNA (specificity)

(v) Repeat all experiments under simulated microgravity to confirm performance consistency

Selin Kocalar, Bess M Miller, Ally Huang, Emily Gleason, Kathryn Martin, Kevin Foley, D Scott Copeland, Michael C Jewett, Ezequiel Alvarez Saavedra, Sebastian Kraves. Validation of cell-free protein synthesis aboard the International Space Station. ACS Synth Biol. 2024 Mar 15;13(3):942-950. doi:10.1021/acssynbio.3c00733