Week 9 HW: Cell Free Systems

Homework Part A: General and Lecturer-Specific Questions

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.
Cell-free protein synthesis (CFPS) offers a fundamentally different operating logic from in vivo expression: because there is no living cell to maintain, the reaction environment is open and directly accessible to the experimenter. This openness translates into three practical advantages. First, reaction components — amino acid concentrations, buffer conditions, redox potential, template concentration — can be tuned independently and in real time without the buffering effects of cellular homeostasis. Second, toxic proteins that would kill or arrest growing cells can be expressed freely in CFPS, since there is no cell viability to protect. Third, non-canonical amino acids, isotopic labels, or synthetic chemical groups can be incorporated site-specifically by supplementing the reaction directly, enabling protein engineering strategies that are impossible to sustain through the protein expression machinery of a living cell. Two cases where cell-free expression is specifically more advantageous than cell-based production are: (1) membrane protein structural studies, where the absence of competing cellular membranes allows co-translational insertion directly into defined lipid nanodiscs of controlled composition, circumventing the protein aggregation and misfolding problems that arise during over-expression in intact cells; and (2) rapid on-demand diagnostic biosensors, where freeze-dried CFPS reactions can be deployed at the point of need without cold-chain infrastructure or biohazard containment — capabilities recently validated aboard the International Space Station.

2. Describe the main components of a cell-free expression system and explain the role of each component.
A cell-free expression system can be understood as a minimal reconstruction of the cellular central dogma pathway. The core components and their roles are as follows. The DNA or mRNA template encodes the protein of interest and acts as the informational input; a strong promoter (e.g., T7) is typically used when a DNA template drives transcription. RNA polymerase (either endogenous in crude lysates or supplied purified as T7 RNAP in PURE systems) transcribes the DNA into mRNA. The ribosome is the catalytic core of translation, reading the mRNA and elongating the polypeptide chain with the assistance of elongation factors (EF-Tu, EF-G) and initiation/release factors; roughly 4 ATP equivalents are consumed per peptide bond formed. Aminoacyl-tRNA synthetases (aaRSs) charge each of the 20 tRNAs with their cognate amino acid, and tRNA molecules deliver those charged amino acids to the ribosome A-site. Amino acids serve as the building block pool; depletion of the amino acid pool is one of the primary causes of reaction stalling in crude lysate systems. The energy regeneration module — commonly phosphoenolpyruvate (PEP) plus pyruvate kinase, creatine phosphate plus creatine kinase, or 3-phosphoglycerate (3-PGA) — continuously regenerates ATP and GTP from ADP/GDP to sustain translation. Magnesium ions are essential cofactors for ribosome function and nucleotide-dependent enzymes; their concentration must be carefully titrated. Potassium ions set the ionic environment required for ribosome activity. Finally, polyethylene glycol (PEG) or similar crowding agents mimic the macromolecular crowding of the cytoplasm and can improve translation efficiency. In the PURE system, all these components are defined and provided as purified proteins, offering reproducibility and the absence of contaminating nucleases and proteases.

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.
Energy regeneration is critical in CFPS because translation is an inherently ATP- and GTP-intensive process — approximately 4 high-energy phosphate equivalents are consumed per amino acid incorporated (2 ATP for aminoacyl-tRNA charging, 1 GTP for tRNA delivery to the ribosome, and 1 GTP for translocation) (Jewett & Swartz, 2004). Without continuous regeneration, the ATP pool is rapidly depleted, causing translation to stall. A further complication is the accumulation of inorganic phosphate (Pi) as a byproduct of phosphotransfer reactions: elevated Pi sequesters Mg²⁺, which is an essential ribosomal cofactor, thereby inhibiting both transcription and translation. An effective energy system must therefore not only regenerate ATP but also limit Pi accumulation (Calhoun & Swartz, 2007). One reliable method is the 3-phosphoglycerate (3-PGA) system, in which 3-PGA enters a truncated glycolytic pathway to regenerate ATP while producing only pyruvate and acetate as by-products — neither of which chelates Mg²⁺ appreciably. Studies have shown that 3-PGA-powered CFPS sustains reactions for several hours and achieves yields exceeding 1 mg/mL of recombinant protein (Kim & Swartz, 2001). A complementary strategy is to use a fed-batch or semi-continuous dialysis reactor format, in which fresh substrates (ATP precursors, amino acids, cofactors) are continuously exchanged into the reaction while inhibitory by-products are dialysed out, extending productive synthesis from hours to potentially days (Spirin et al., 1988). For classroom or field-deployable settings, the simpler creatine phosphate / creatine kinase (CP/CK) system remains widely used, despite the 1:1 stoichiometric phosphate release it entails, because of its low cost and ease of formulation.

4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
Prokaryotic CFPS systems — most commonly derived from E. coli lysates — are fast to prepare, inexpensive, highly productive (yields of 1–4 mg/mL are achievable in optimised formats), and compatible with a wide range of T7-based expression vectors. Their principal limitation is the absence of the eukaryotic post-translational modification machinery: E. coli extracts cannot perform N-linked glycosylation, and the reducing cytoplasmic environment is unfavourable for the formation of disulfide bonds, which are essential for many human therapeutic proteins. Eukaryotic CFPS systems — including wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and Chinese hamster ovary (CHO) cell lysates — provide access to chaperones, signal recognition particles, and post-translational processing machinery that support proper folding of complex human proteins. They tend to be slower and more expensive than prokaryotic systems, but are indispensable when the target protein requires glycosylation, specific disulfide connectivity, or processing by signal peptidase. For the prokaryotic system (E. coli extract), an excellent choice is single-chain variable fragment (scFv) antibody, a small (~27 kDa) recombinant antibody format that does not require glycosylation and whose binding function can be verified rapidly by an ELISA-based assay. The fast turnaround of bacterial CFPS (reactions complete within 4–6 hours) is ideal for iterative screening of antibody variants during affinity maturation campaigns. For the eukaryotic system (CHO or insect cell extract), erythropoietin (EPO) is the appropriate choice. EPO is a 165-amino acid glycoprotein hormone in which three N-linked and one O-linked glycan chains account for approximately 40% of its molecular weight and are critical for its in vivo half-life and receptor-binding activity. Expressing EPO in a prokaryotic system yields aglycosylated protein with substantially reduced biological activity; a CHO-based CFPS system that includes microsomes or glycosylation enzymes can produce a glycoform closer to the therapeutic molecule.

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.
Membrane proteins (MPs) represent the most challenging class of targets for CFPS because their hydrophobic transmembrane domains are insoluble in aqueous solution: without a lipid environment, they aggregate irreversibly into inclusion body-like precipitates immediately after synthesis. A well-designed cell-free membrane protein experiment must therefore couple protein synthesis to a compatible hydrophobic scaffold present in the reaction from the outset. The recommended strategy is co-translational insertion into pre-formed nanodiscs. Nanodiscs are discoidal phospholipid bilayer patches stabilised by an amphipathic membrane scaffold protein (MSP); their diameter (~10 nm) and lipid composition can be controlled precisely. By including nanodiscs at optimised concentrations (typically 0.2–2 mg/mL) in the CFPS reaction, the nascent transmembrane protein can fold co-translationally into the bilayer rather than encountering aqueous solution at all, preserving its native fold and function. Studies have shown that nanodisc-based CFPS supports correct folding of GPCRs, ion channels, and multi-pass transporters at yields sufficient for structural studies by NMR or cryo-EM. Three specific challenges and how to address them: (1) Aggregation during synthesis — mitigated by using lipid nanodiscs as described above, supplemented if needed with detergents at sub-CMC concentrations such as Brij-35 to stabilise partially-folded intermediates; (2) Low expression yield — membrane proteins are often toxic in in-vivo systems but in CFPS this is no constraint; yield can be maximised by optimising the concentration of nanodiscs, adjusting Mg²⁺ levels (often 10–14 mM for membrane protein CFPS rather than the standard 8–10 mM), and screening N-terminal fusion tags to improve ribosome engagement; (3) Verification of correct folding — since Western blotting confirms synthesis but not function, activity assays (e.g., ligand binding ELISA for GPCRs, patch-clamp for channels) or limited proteolysis footprinting should be used to confirm the protein has adopted its native architecture.

6. 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.
Low yield in a CFPS reaction can arise at multiple points in the expression pathway. Here are three common causes and their corresponding troubleshooting strategies. Reason 1 — Premature energy depletion and ATP starvation. If the energy regeneration system is insufficient or the secondary energy source (e.g., phosphoenolpyruvate) is consumed too quickly, ATP levels drop below the threshold required to sustain elongation, causing ribosomes to stall prematurely. The troubleshooting strategy is to measure the reaction’s pH over time using a microelectrode or pH-sensitive dye (acidification indicates Pi accumulation and ATP exhaustion) and to switch to a more sustained energy substrate such as 3-PGA, which produces less Pi per ATP regenerated, or to implement a fed-batch format with controlled substrate addition. Reason 2 — mRNA instability and degradation. Crude cell extracts contain residual ribonucleases that can degrade the mRNA template, especially if it lacks a strong 5’ untranslated region (UTR), a stable secondary structure at the 3’ end, or is not capped in eukaryotic systems. The troubleshooting strategy is to run the reaction without protein expression template and assess background RNase activity using a fluorescent RNA reporter; if high, add RNase inhibitor (e.g., RNasin), switch to a DNA template with a strong ribosome binding site, or use a PURE system that is free of nucleases. Reason 3 — Suboptimal magnesium and potassium ion concentrations. Both Mg²⁺ and K⁺ profoundly affect ribosome assembly and activity, and their optimal concentrations depend on the extract lot, target protein, and energy system used. A single mM deviation in [Mg²⁺] can halve protein yield. The troubleshooting strategy is to perform a systematic two-dimensional titration of Mg²⁺ (range: 4–16 mM) and K⁺ (range: 60–200 mM) against protein yield measured by fluorescence (if GFP is used as a reporter) or SDS-PAGE densitometry, and re-optimise for each new extract batch or protein target.

Homework question from Kate Adamala

Design an example of a useful synthetic minimal cell as follows:

1. Pick a function and describe it.
   a. What would your synthetic cell do? What is the input and what is the output?
   The proposed synthetic minimal cell (SMC) functions as a field-deployable water quality sensor for antibiotic resistance. The input is the presence of beta-lactam antibiotic residues (specifically ampicillin) in environmental water samples, detected via a riboswitch aptamer domain that undergoes a conformational change upon ligand binding. The output is fluorescent GFP produced by the encapsulated CFPS system, reportable visually with a handheld fluorescence viewer such as the miniPCR P51 Molecular Fluorescence Viewer.
   b. Could this function be realized by cell-free Tx/Tl alone, without encapsulation?
   No. Encapsulation is essential for two reasons: first, the lipid membrane creates a concentration gradient that amplifies the input signal — only molecules that enter or diffuse across the bilayer trigger the sensor, reducing false positives from trace non-specific binding. Second, the membrane physically separates the CFPS machinery from environmental nucleases and proteases present in raw water samples, which would otherwise degrade the RNA aptamer and mRNA templates. Without encapsulation, the reaction would be rapidly inactivated in complex environmental matrices.
   c. Could this function be realized by genetically modified natural cell?
   Yes, in principle: an E. coli strain engineered with an ampicillin-responsive transcription factor driving GFP expression could detect beta-lactams. However, release of live GMO bacteria into environmental water samples raises serious biosafety and ecological concerns, and the engineered organism may not survive or function predictably in the field. The SMC offers a fully abiotic, self-contained, containable alternative with no replication capacity.
   d. Describe the desired outcome of your synthetic cell operation.
   In the presence of ampicillin above a defined threshold concentration (~10 µM), the riboswitch aptamer within the SMC adopts its ligand-bound conformation, allowing ribosomal readthrough of an upstream inhibitory sequence and enabling translation of the GFP reporter. The operator observes green fluorescence from the SMC population when viewed under blue LED excitation — a simple positive/negative readout of water contamination.

2. Design all components that would need to be part of your synthetic cell.
   a. What would be the membrane made of?
   POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) as the primary structural lipid, with 10 mol% POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)) to introduce a slight anionic character that improves vesicle stability and reduces aggregation. No cholesterol is required for this room-temperature sensor application.
   b. What would you encapsulate inside? Enzymes, small molecules.
   A bacterial cell-free Tx/Tl system (E. coli S30 extract), the riboswitch-GFP DNA construct, an ATP regeneration module (creatine phosphate + creatine kinase), all 20 amino acids, NTPs, Mg²⁺, and K⁺ at optimised concentrations.
   c. Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason? (hint: for example, if you want to use small molecule modulated promotors, like Tet-ON, you need mammalian)
   Bacterial (E. coli S30 extract), because the riboswitch is derived from a prokaryotic aptazyme architecture and functions via modulation of ribosome access to the Shine-Dalgarno sequence — a mechanism specific to bacterial translation.
   d. How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)
   Ampicillin is a small, moderately amphipathic beta-lactam molecule (~349 Da) that can passively permeate phospholipid bilayers to a limited but measurable extent. At the ampicillin concentrations relevant for contamination detection (10–100 µM), passive permeation is sufficient to trigger the internal riboswitch without requiring an active transporter. GFP output remains internal and is detected non-destructively by fluorescence spectroscopy or imaging.

3. Experimental details
   a. List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)
   Lipids: POPC (Avanti Polar Lipids #850457), POPG (Avanti Polar Lipids #840457)
   Genes: Ampicillin-responsive riboswitch–GFP fusion: a synthetic construct encoding an engineered aptazyme responsive to beta-lactams (based on the aptazyme architecture of Wieland & Hartig, 2008) fused upstream of a GFP ORF under a T7 promoter
   Specific gene-GFP variant: sfGFP (superfolder GFP; Addgene plasmid #54579), chosen for its robust folding kinetics in cell-free systems
   b. How will you measure the function of your system?
   Measure GFP fluorescence of the SMC suspension using a plate reader (excitation 488 nm, emission 510 nm) or a P51 handheld viewer for field deployment. A positive control containing a constitutively expressed GFP plasmid and a negative control of vesicles containing a scrambled riboswitch should bracket every experiment. Vesicle integrity is confirmed by dynamic light scattering (DLS) before and after the assay.

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.
A freeze-dried cell-free biosensor woven directly into a protective work garment that changes from orange to green fluorescence within 90 minutes of contact with airborne organophosphate pesticide residues, providing farm workers with a passive, wearable early-warning system for chemical exposure.

How will the idea work, in more detail? Write 3-4 sentences or more.
The garment integrates freeze-dried cell-free (FDCF) synthetic biology circuits embedded in cellulose-based reaction insets woven into the chest panel of the fabric, using the methodology developed by Nguyen, Soenksen et al. The FDCF reaction encodes an organophosphate-responsive genetic circuit: acetylcholinesterase (AChE) activity is coupled to a split-reporter system such that AChE inhibition by organophosphates — detectable at concentrations as low as 10 nM — derepresses expression of a fluorescent aptamer. A polymeric optical fibre network interwoven with the fabric continuously probes each reaction zone for changes in fluorescence (orange baseline → green signal-positive), and the output is transmitted via Bluetooth to a paired smartphone application, alerting the wearer of exposure in real time. The reaction chambers are hermetically sealed and activated only by moisture — either sweat or rain — contacting the fibre insets, preventing premature activation during storage.

What societal challenge or market need will this address?
Organophosphate pesticide exposure is the leading cause of acute agricultural poisoning worldwide, responsible for an estimated 385 million cases of unintentional acute pesticide poisoning per year (WHO, 2019). Farm workers in low-to-middle income countries frequently lack access to personal air quality monitors or laboratory testing infrastructure. A textile-embedded FDCF sensor worn as ordinary work clothing would provide continuous, real-time, instrument-free exposure monitoring, enabling workers to evacuate contaminated areas before symptoms manifest and generating timestamped exposure logs usable in occupational health assessments.

How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?
The three primary limitations — activation with water, stability in humid environments, and one-time use — are addressed as follows. Activation by water is an inherent design feature here rather than a drawback: the sensor is intentionally triggered by sweat contact, and the fabric’s hydrophobic outer layer acts as a moisture gate, ensuring activation only after meaningful liquid contact. Long-term stability is achieved by lyophilising the CFPS reactions in the presence of trehalose as a cryoprotectant and sealing individual reaction zones in a vapour-barrier polymer matrix; prior work has demonstrated FDCF stability at ambient temperature for at least six months under these conditions. The single-use constraint is addressed architecturally: reaction zones are modular insets that can be removed and replaced by the wearer after each work day, analogous to replacing a spent filter cartridge, while the fibre optic network and smartphone interface are reusable across many cycles.

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)
Galactic cosmic radiation (GCR) and solar energetic particles present a significant health risk during deep-space missions, inducing DNA double-strand breaks (DSBs) and oxidative base damage in astronaut cells. Current biomonitoring of radiation-induced DNA damage aboard the ISS requires blood draws, cryopreservation, and Earth-based laboratory analysis — an impractical pipeline for future lunar or Mars missions where resupply is impossible. Developing a rapid, portable, crew-operable assay for real-time radiation exposure biomonitoring is critical to protect astronaut health and to inform mission planning and shielding design for exploration beyond low-Earth orbit.

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) Target: p21 (CDKN1A) mRNA — a transcriptional target of the p53 DNA damage response pathway, reliably upregulated within hours of ionising radiation exposure in human cells.

3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)
When ionising radiation causes DNA DSBs, the tumour suppressor p53 is activated and drives transcription of p21/CDKN1A, a cyclin-dependent kinase inhibitor that halts the cell cycle to allow DNA repair. Because p21 mRNA accumulates in cells proportionally to the absorbed radiation dose, it is a well-validated molecular dosimeter. Importantly, p21 mRNA can be extracted from crew saliva or buccal cells — a non-invasive sample type fully compatible with spaceflight constraints — and detected using the BioBits® toehold switch platform without the need for PCR equipment or cold-chain reagents.
4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words) Hypothesis: BioBits®-based toehold switch sensors designed to detect human p21 mRNA will produce a fluorescent readout proportional to radiation dose, as measured in buccal cell RNA extracts collected from astronauts aboard the ISS, and will perform comparably to Earth-based qRT-PCR reference measurements. Reasoning: Toehold switches — linear RNA hairpin structures that undergo conformational change upon hybridisation to a complementary trigger RNA — have been validated as highly sensitive, sequence-specific nucleic acid sensors in cell-free systems with detection limits as low as picomolar concentrations . Prior work by Kocalar et al. (2024) demonstrated that BioBits® performs robustly in microgravity. Because p21 mRNA is a human transcript expressed in cells easily obtainable by non-invasive buccal swab, the assay requires no genetic engineering of the crew, preserves biosafety, and is activatable by simple rehydration of the lyophilised BioBits® pellet with the extracted RNA sample.

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)
Samples: Buccal swabs collected from crew members at three time points — pre-mission baseline, 72 hours after a known solar energetic particle (SEP) event (using ISS radiation dosimetry logs as the reference), and at mission end. RNA is extracted using a portable lysis buffer compatible with the miniPCR kit. Experiment: p21 toehold switch BioBits® reactions are rehydrated with crew RNA extract and incubated for 60 minutes at 37 °C. Fluorescence is read using the P51 Molecular Fluorescence Viewer; image intensity is quantified via the paired smartphone app. Controls: Non-irradiated Earth buccal RNA (negative control); synthetic p21 mRNA spike-in (positive control); scrambled-sequence toehold switch (specificity control).