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 superior flexibility and control compared to in vivo methods. You can directly add DNA templates, tweak reaction conditions like temperature or pH on the fly, and incorporate non-natural amino acids without genetic engineering, which is impossible in living cells due to their complex regulation. It also allows real-time monitoring and manipulation of variables like cofactor concentrations.

CFPS is better in two cases: (1) toxic proteins, like bacterial toxins that kill host cells during expression; (2) membrane proteins, where cell-free systems use nanodiscs or liposomes to mimic membranes without cellular toxicity or folding issues.

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

A typical CFPS systemhas:

-DNA template: Encodes the target protein; often under a strong promoter like T7. -Ribosomes: Translate mRNA into protein; sourced from crude cell lysates. -RNA polymerase (T7 RNA pol), NTPs for mRNA synthesis. -Other proteins you need -Translation factors: Aminoacyl-tRNA synthetases, elongation/initiation factors to charge tRNAs and assemble proteins. -Energy sources: ATP, GTP, creatine phosphate. -Amino acids and cofactors: Building blocks and Mg²⁺/K⁺ ions for stability. -Lysate: Crude extract providing most components.

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 because CFPS rapidly consumes ATP/GTP, halting reactions within minutes without replenishment, unlike cells with metabolic pathways.

A phosphoenolpyruvate (PEP)/pyruvate kinase system: PEP donates phosphate to ADP via kinase, continuously regenerating ATP. Add 20-50 mM PEP and 0.1 mg/mL pyruvate kinase to your reaction mix.

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

I would produce GFP in prokaryotic CFPS, it’s prokaryotic-optimized, high-yield, no glycosylation needed. I would produce a glycoprotein like erythropoietin in eukaryotic CFPS for proper N-glycosylation essential for activity.

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: Membrane proteins aggregate without lipids, misfold, or insert poorly.

Design:Use a lipid-detergent mix (1% Brij-58 + POPC liposomes) or nanodiscs (MSP1D1 belt proteins + lipids).Co-express with chaperones (Skp) from a polycistronic template.Optimize Mg²⁺ (10-15 mM) and temperature (25°C) for insertion.Test via fluorescence (GFP) or functional assays ( transport activity).This solubilizes proteins during synthesis, boosting yield 10-fold.

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.

Reason 1: Inhibitory DNA impurities =>Strategy: Purify plasmid with column kits and test dilutions. Reason 2: Suboptimal energy depletion =>Strategy: Add fresh PEP/GTP mix and monitor ATP via luciferase assay. Reason 3: Protease degradation => Strategy: Use protease-deficient lysates and add inhibitors like PMSF.

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?

Function: Detect arsenic in water and signal via fluorescence. Input: arsenite . Output: fluorescent resorufin.

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

No, it needs encapsulation for controlled release and membrane signaling.

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

      Yes (E. coli with ars operon), but synthetic version allows modularity for sensors without genomic integration.

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

Synthetic cells glow pink near toxic arsenic levels, enabling visual water safety checks.

2.Design all components that would need to be part of your synthetic cell.

What would be the membrane made of?

POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) + 20% cholesterol liposomes.

What would you encapsulate inside? Enzymes, small molecules.

Bacterial (E. coli) Tx/Tl lysate, 1 mM D-luciferin, DNA for ArsR-repressed aHL (alpha-hemolysin) pore and luciferase under Ars promoter.

Which organism your Tx/Tl system will come from? Is bacterial OK, or do you need a mammalian system for some reason?

Bacterial (E. coli), ArsR riboswitch is prokaryotic

How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

Arsenite permeates membrane passively; binds ArsR, induces aHL expression for luciferin efflux, activating external luciferase.

3.Experimental details

a.List all lipids and genes.

-Lipids: POPC, cholesterol. -Genes: arsR (repressor, UniProt P0A1C7), aHL (hlyA from S. aureus, UniProt P0DQI1), luciferase (luxA from Vibrio fischeri). -Biological cells: None

b.How will you measure the function of your system?

Fluorescence plate reader (560 nm excitation) for resorufin, dose-response curve vs. arsenite.

Homework question from Peter Nguyen

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

Smart fabric-embedded freeze-dried cell-free sensors that change color in response to sweat pH, alerting wearers to dehydration.

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

Freeze-dry E. coli CFPS with pH-sensitive GFP plasmid and pH indicator dyes into cotton fibers via lyophilization-spray coating. Water from sweat rehydrates the mix, triggering protein expression, acidic pH shifts GFP fluorescence from green to yellow, visible under UV or as embedded dye release.

3.What societal challenge or market need will this address?

Addresses dehydration and heat stress for athletes, outdoor workers, and other people exposed to Romania’s increasingly frequent heatwaves.

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

Stability via trehalose cryoprotectant (extends shelf-life 1+ year) or one-time use mitigated by modular reloading or multi-compartment fabrics; water activation natural from body moisture.

Homework question from Ally Huang

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.

Microgravity impairs plant growth on long missions, reducing food and oxygen because of the weakened roots and altered gene expression (EXPANSIN genes). Vital for Mars missions needing sustainable hydroponics, scientifically intriguing as it reveals gravity’s role in auxin signaling and cell wall remodeling,BioBits CFPS enables rapid, resource-light testing in space.

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.

EXP1 expansin gene from Arabidopsis thaliana.

3.Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses.

EXP1 loosens cell walls for root elongation; microgravity downregulates it, stunting growth. Studying EXP1 expression in CFPS mimics space conditions), linking to pathways disrupted off-Earth, and informing genetic fixes for space crops.

4.learly state your hypothesis or research goal and explain the reasoning behind it.

Overexpression of EXP1 in BioBits CFPS will restore simulated microgravity-impaired root growth signals, as measured by downstream fluorescent reporters. Reasoning: Earth-based studies show EXP1 compensates gravity loss in clinostats; CFPS bypasses live plant limits in space, allowing direct gene dosing. Hypothesis tests if EXP1 alone boosts growth factors without full cellular contex

https://pmc.ncbi.nlm.nih.gov/articles/PMC9937981/

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