Week 9 HW: 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.

The first advantage of cell-free protein synthesis (CFPS) over traditional in vivo methods is that it avoids the ethical concerns associated with modifying living cells; CFPS uses cellular machinery such as ribosomes and enzymes. The second advantage of CFPS is the time and cost. CFPS takes around 1-2 hours while cell-based expressions, such as in E. coli, take around a few days to a week, depending on the expression. Third, there is increased biosafety and controllability with CFPS. CFPS are more controllable and programmable. Unlike using living cells which can escape into the environment and potentially cause harm depending on the organism. Lastly

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

A cell-free expression system contains the molecular machinery for transcription and translation containing ribosomes to build the target protein, tRNA to bring amino acids to the target site, RNA polymerase to transcribe DNA into RNA, and translation factors to elongate a DNA template to encode the target protein. The DNA template, usually a plasmid or linear DNA, instructs the cell-free system to produce the protein. The template includes a promoter, a coding sequence, and a terminator. All 20 amino acids are also included. The energy system of the cell, such as ATP to power transcription, and GTP to power translation. Finally and most importantly, the cell-free system’s “environment” is made up of Mg²⁺, K⁺ to stabilise ribosomes and enzymes, NTPs (ATP, CTP, GTP, UTP), for mRNA synthesis, and buffers to maintain pH level, for the reactions to occur efficiently and successfully.

3. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. 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.

To optimise a membrane protein in cell-free expression, I would first choose the lysate based on the protein type: E. coli extract for a bacterial target, and wheat germ or another eukaryotic extract for an eukaryotic target. I would then screen several membrane mimics directly in the reaction—such as mild detergents, liposomes, and nanodiscs—to promote cotranslational insertion and reduce aggregation. I would test multiple conditions for magnesium, temperature, DNA concentration, and reaction format, then compare total expression, soluble fraction, and functional activity rather than yield alone. The major challenges are aggregation, misfolding, poor membrane insertion, and low functional recovery, and these are addressed by matching the CFPS chassis to the target, providing an appropriate lipid environment during synthesis, and screening conditions systematically.

4. 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.

The first reason for a low yield in the target protein is an imbalance in the composition as well as a lack of ATP and GTP. Cell-free systems rely on ATP, GTP, amino acids, salts, cofactors, and an appropriate magnesium and potassium balance. To troubleshoot, one uses the correct measuring tools and is exact in optimising each component especially magnessium and template concentration. One must also ensure the correct temperatures are used with measured time. One can also switch energy usages as cell free systems rely on massive amounts of ATP and GTP. One could use Creatine Phosphate or Glucose.

The second reason for a low yield is inefficient folding and aggregation. In a cell-free extract, proteins can become stuck together, forming inclusion bodies rather than functional proteins. This could be due to a higher concetration in protein. To fix this, one could supplement the mix with purified DnaK/J-GrpE or GroEL/ES complexes to assist with folding. Dropping the incubation temperature can also help. Slower translation often helps the protein find its native conformation.

The final reason for a low yield of the target protein in template degradation is also commonly known as the RNase problem. Cell-free extracts contain endogenous nucleases. DNA template or newly transcribed mRNA is digested before ribosomes can finish their run, and yield drops completely in the first 30-60 seconds, even if the system has enough energy. One can supplement the reaction with 0.5–1.0 U/μL of a recombinant RNase inhibitor. There are also methods to protect linear DNA ends from degradation by the RecBCD exonuclease, such as adding the GamS protein to PCR products. One should also ensure there is no contamination in the tools to minimise surface-bound nuclease contamination.

Homework question from Kate Adamala

Part 1

Q1. What would your synthetic cell do? What is the input and what is the output?

The synthetic cell functions as a biological electromagnetic field sensor. hCRY2 contains a FAD cofactor that, upon blue light absorption at approximately 450 nm, forms a radical pair whose singlet/triplet spin state ratio is altered by external magnetic fields. This change in spin state modulates hCRY2 repressor activity, which drives expression of a nanoLuc bioluminescence reporter. The two inputs are blue light, which initiates radical pair formation, and magnetic field strength and orientation, which modulate the radical pair spin chemistry. The output is nanoLuc luminescence, whose intensity is inversely proportional to hCRY2 repressor activity and therefore encodes the magnetic field experienced by the cell.

Q2. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?

No, encapsulation is essential. In bulk solution, radical pairs are rapidly quenched by molecular oxygen and random collisions, making magnetic sensitivity impossible. Encapsulation provides an oxygen-scavenged microenvironment that extends radical pair lifetime long enough for the magnetic field to exert its effect. It also maintains the local protein concentrations and molecular crowding needed for correct spin chemistry, and provides a stable compartment to sustain the Tx/Tl machinery required for the reporter output.

Q3. Could this function be realized by a genetically modified natural cell?

In principle yes, since hCRY2 is already expressed in human cells as part of the circadian clock. However, endogenous antioxidants would quench radical pairs unpredictably, intracellular oxygen and FAD levels cannot be independently controlled, and the magnetosensing signal cannot be isolated from hCRY2’s other cellular functions. The synthetic cell provides full control over every parameter governing the radical pair mechanism, making it the more precise and tractable approach.

Q4. Describe the desired outcome of your synthetic cell operation.

The desired outcome is a population of GUVs that produce measurably different nanoLuc luminescence levels depending on the strength and orientation of an applied magnetic field, under blue light illumination. The response should be reversible upon removal of the field, and should be completely abolished when FAD is omitted or when the internal oxygen scavenging system is disabled, confirming that the output is mechanistically dependent on radical pair spin chemistry.

Part 2 — Component Design

2.1 What would the membrane be made of?

The membrane would consist of GUVs composed of DPPC at 50 mol%, DOPE at 30 mol%, and cholesterol at 20 mol%. DPPC’s saturated acyl tails and the high cholesterol content produce a low-fluidity, low-oxygen-permeability bilayer that is specifically chosen to extend the radical pair lifetime inside the vesicle by limiting oxygen ingress.

2.2 What would you encapsulate inside?

The interior would contain purified hCRY2 protein pre-loaded with FAD as the magnetosensor, free FAD at 150 µM, plasmids encoding AmCLK, AmCYC, and nanoLuc under E-box promoter control, and a mammalian cell-free extract to provide Tx/Tl machinery. An oxygen scavenging system of glucose, glucose oxidase, and catalase would protect the radical pair, while Trolox at 1 mM would stabilize its lifetime, and creatine phosphate with creatine kinase would regenerate ATP.

2.3 Which organism will the Tx/Tl system come from?

A mammalian cell-free extract from HEK293 or HeLa cells is required. A bacterial system cannot be used because the E-box promoter driving nanoLuc requires eukaryotic RNA polymerase II machinery, hCRY2 signaling depends on eukaryotic post-translational modifications such as phosphorylation by casein kinase 1 epsilon, and proper FAD incorporation requires eukaryotic chaperones. A commercially available system such as the Thermo Fisher 1-Step Human IVT kit is appropriate and is additionally well-matched to hCRY2 given its human origin.

2.4 How will your synthetic cell communicate with the environment?

No membrane channels are required. Blue light photons and the magnetic field both pass freely through the lipid bilayer and act directly on the encapsulated hCRY2-FAD complex, and nanoLuc bioluminescence is emitted as photons that pass outward through the membrane to be detected externally. The entire input/output communication of this synthetic cell is electromagnetic and requires no membrane transport proteins.

Part 3 — Experimental Details

3.1 List of all lipids and genes

The lipids required are DPPC (Avanti 850355), DOPE (Avanti 850725), and cholesterol (Sigma C8667). The genes required are hCRY2 (Gene ID 4236) as the magnetosensor, AmCLK (Gene ID 406114) and AmCYC (Gene ID 411055) as the transcriptional activators, and nanoLuc (Promega) as the reporter, all codon-optimized for mammalian expression. Key small molecules include FAD at 150 µM (Sigma F6625), glucose with glucose oxidase and catalase for oxygen scavenging, Trolox at 1 mM (Sigma 238813), and creatine phosphate with creatine kinase for ATP regeneration.

3.2 How will you measure the function?

Vesicles would be exposed to magnetic fields of 0 to 500 µT via a Helmholtz coil array while nanoLuc luminescence is recorded on a plate reader, to determine whether output scales with field strength. Field orientation would be rotated across all three axes at fixed strength to test directional sensitivity. The critical mechanistic control is the RF oscillating field test, in which a radiofrequency field applied at the Larmor frequency disrupts radical pair spin state interconversion and should abolish the magnetic response, confirming a quantum mechanical origin. FAD-minus and oxygen scavenging-minus conditions would serve as additional controls confirming cofactor and radical pair dependence respectively.

Homework question from Peter Nguyen

Freeze-Dried Cell-Free Systems in Textiles Application Field: Textiles and Fashion

One-sentence pitch:

A garment embedded with freeze-dried cell-free biosensors that activates upon contact with the wearer’s sweat, translating electrodermal skin conductivity into a shifting visual patina on the textile surface that externalises the wearer’s neurological and emotional state in real time.

How will it work?

Freeze-dried cell-free systems remain entirely dormant when dry and reactivate upon hydration, meaning the wearer’s own sweat serves as the biological trigger without any external power or living cells required. The textile is embedded with microreactors containing freeze-dried Tx/Tl machinery programmed to respond to biomarkers present in sweat, specifically the ionic composition and conductivity changes associated with electrodermal activity, which is a direct readout of sympathetic nervous system arousal. Upon activation, the cell-free system drives expression of chromogenic enzymes or pigment-producing biosynthetic pathways, such as the violacein pathway, that physically alter the textile’s colour and patterning in proportion to the intensity of the electrodermal signal. The result is a dynamic, personalised patina that is unique to each wearer and each emotional experience, forming a wearable record of one’s inner neurological world rendered visibly on the body.

What societal challenge or market need does this address?

Mental health and emotional experience remain largely invisible and difficult to communicate, both to others and to the individual themselves. This concept addresses the growing cultural need for tools that make inner states legible and expressible, sitting at the intersection of mental health awareness, biofeedback wearables, and sustainable fashion. Unlike electronic biosensors, this system requires no battery, no screen, and no data transmission, making it radically accessible, low-cost, and free from the privacy concerns associated with digital health monitoring. It also responds to the fashion industry’s interest in programmable and living materials as an alternative to static, environmentally costly textile production.

How will you address the limitations of cell-free reactions?

The primary limitations of freeze-dried cell-free systems are their one-time activation, finite reagent supply, and sensitivity to environmental conditions. The one-time use constraint is reframed here as a design feature rather than a drawback, since each wearing produces a permanently fixed, unrepeatable patina that functions as a record of that specific emotional experience, giving the garment biographical value over time. Stability during storage and transport is addressed by the freeze-drying process itself, which has already been demonstrated to preserve cell-free system activity across months without refrigeration when embedded in paper and textile substrates. To extend functional longevity across multiple wears, microreactor chambers within the textile can be designed as replaceable or rechargeable modules, allowing the biological sensing layer to be refreshed while the garment itself is reused, addressing both sustainability and repeatability.

Homework Questions From Ally Huang

Genes in Space Mock Proposal BioBits Cell-Free Lichen-Inspired Radiation Biosensor for Astronaut Health Monitoring

Background

Astronauts on long-duration missions are exposed to cosmic radiation approximately 200 times more intense than on Earth, generating reactive oxygen species that damage DNA and increase cancer risk. Lichen are among the only organisms demonstrated to survive direct exposure to open space, enduring vacuum, unfiltered cosmic radiation, and extreme temperature swings during ESA’s EXPOSE experiments on the ISS. Their cyanobacterial partner Nostoc has evolved highly efficient oxidative stress response systems fine-tuned for exactly these conditions. Harnessing this space-proven biological machinery as a cell-free diagnostic could provide astronauts with a lightweight, resource-minimal biosensor uniquely suited to the demands of deep space exploration.

Molecular Target

The molecular target is the OxyR oxidative stress sensor protein and its downstream KatG promoter, derived from the lichen cyanobacterial partner Nostoc, driving fluorescent reporter expression in BioBits.

How the Target Relates to the Challenge

In Nostoc, reactive oxygen species generated by radiation directly oxidise the OxyR protein, causing a conformational change that activates the KatG promoter and upregulates antioxidant defence genes. By placing a fluorescent reporter gene downstream of the KatG promoter within the BioBits cell-free system, ROS present in an astronaut’s saliva sample can activate this space-proven stress circuit, producing a fluorescent signal proportional to oxidative stress levels. Unlike the equivalent E. coli system, OxyR and KatG were shaped by evolution in an organism that genuinely survives space conditions, making this a more robust and contextually appropriate sensing circuit.

Hypothesis and Reasoning

We hypothesise that a freeze-dried BioBits cell-free system incorporating Nostoc OxyR as the sensor protein and a KatG-driven fluorescent reporter will produce a measurable, dose-dependent fluorescent signal when rehydrated with saliva samples from astronauts experiencing radiation-induced oxidative stress. We further hypothesise that the addition of usnic acid, a lichen-derived secondary metabolite with known UV-absorbing and antioxidant properties, to the freeze-dried disc formulation will improve system stability and longevity under space radiation conditions compared to a standard BioBits formulation. The reasoning is that Nostoc evolved its OxyR/KatG circuit under conditions of genuine radiation stress, making it more sensitive and robust than equivalent circuits from non-space-adapted bacteria. Usnic acid, which lichen use to shield their photosynthetic partners from radiation damage, could perform the same protective role for the cell-free machinery during storage aboard the ISS.

Experimental Plan

Saliva samples collected from astronauts at regular mission intervals would be pipetted onto freeze-dried BioBits® discs containing Nostoc OxyR protein and a KatG-GFP reporter construct, with usnic acid incorporated into the disc matrix. After 30 minutes of incubation, fluorescence would be visualised using the P51 Molecular Fluorescence Viewer. Controls would include discs rehydrated with water alone as a negative control, discs rehydrated with defined hydrogen peroxide solution as a positive ROS control, and discs formulated without usnic acid to quantify its protective contribution to system stability. Ground-based saliva from non-irradiated individuals would serve as baseline reference.