Week 9 HW: Cell Free Systems

This page tackles all homeworks of week 9.

General homework questions

  • 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 systems offer direct access to the reaction environment without a protective, living cell membrane barrier. This allows precise manipulation of chemical variables—such as adjusting pH, tweaking redox potentials, and adding non-canonical amino acids or toxic chemical inhibitors—without killing a host organism.

    • Case 1: Production of highly toxic proteins (e.g., antimicrobial peptides or cytotoxic enzymes) that would kill living E. coli hosts.

    • Case 2: Rapid, high-throughput screening of massive variant libraries where transforming and culturing live cells takes too much time.

    • Case 3: When experimenting on systems that are not all found within a single cell, like testing plant chloroplasts on human skin cells for biohackers (or other ethical sci-fi topics allowing human sensory enhancements, including quantum electromagnetic sensings, which many global presenters talked about).

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

    • Cellular Extract: Crude cytoplasmic lysate (usually from E. coli, wheat germ, or CHO cells) containing ribosomes, aminoacyl-tRNA synthetases, and endogenous translation factors.

    • DNA Template: Plasmids or linear PCR products encoding the target gene under an appropriate promoter (e.g., T7).

    • Energy Mix & Substrates: A mixture of dNTPs/NTPs, amino acids, and vital salts (Mg++, K+) to feed transcription and translation.

    • Energy Regeneration System: High-energy secondary substrates (like phosphoenolpyruvate or creatine phosphate) used to continually replenish ATP supplies.

  • 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.

    • Protein synthesis is thermodynamically expensive; each peptide bond consumes multiple high-energy phosphate bonds. Without a living metabolic network to generate power, free ATP pools deplete in minutes due to rapid hydrolysis and accumulation of inhibitory inorganic phosphate. To ensure a continuous supply, you can use a PEP-pyruvate kinase system where phosphoenolpyruvate (PEP) acts as a high-energy donor, allowing a recycling enzyme to continuously re-phosphorylate spent ADP back into active ATP.

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

    • Prokaryotic systems (like E. coli TX-TL) are high-yield, cheap, and fast, but they lack complex post-translational modifications (PTMs) like glycosylation or proper disulfide bonding. Eukaryotic systems (like HeLa or CHO lysates) are slower and yield less total protein, but they have native endoplasmic reticulum/Golgi machinery for complex folding.

    • Prokaryotic Choice: Green Fluorescent Protein (GFP), because it requires no glycosylation or complex mammalian chaperone folding, making it ideal for cheap, massive yields in E. coli lysates.

    • Eukaryotic Choice: Human Erythropoietin (EPO), because it requires intricate sialic acid glycosylation to function biologically, which prokaryotic machinery cannot perform.

    • Photosynthesis pathway may be mimicked within cell-free systems to allow novel space structures for future exoplanet exploration by evolved humans.

  • 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.

    • The Challenge: Membrane proteins are highly hydrophobic and will rapidly misfold, aggregate, and precipitate out of solution when translated into a watery, open extract without a structural lipid bilayer to shield them.

    The Solution: Supplement the cell-free reaction matrix with synthetic nanodiscs or detergent micelles (like Triton X-100 or DDM). As the ribosome synthesizes the hydrophobic domains, the proteins can spontaneously insert directly into these soluble lipid structures, maintaining proper folding and activity.

    Actually, membranes can also be designed (or generated) such that certain specific nutrients or other solutions are encapsulated within spherical membranes and put inside the cell-free systems. Later, after some time, these spherical membranes can be removed to observe how the cell interacts with the internal components of the membrane, which are designed to mimic the external world. This system may actually invert the topology such that the cell-free system acts like the interior of the cell and the region within the spherical membrane-bound structures is actually made to represent the outside of he cell. (This inverts the inside-out cellular topology!)

  • 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.

    1. Reason: Rapid depletion of the energy source.
      Fix: Supplement the reaction with an improved, higher-capacity secondary energy system like creatine phosphate/creatine kinase.

    2. Reason: High endogenous nuclease/protease activity degrading your DNA template or target protein.
      Fix: Add an RNase inhibitor cocktail or switch to a commercial extract optimized with protease gene knockouts (like a $\Delta rne$ or $\Delta lon/\Delta ompT$ strain).

    3. Reason: Inefficient codon usage slowing down translation.
      Fix: Perform codon optimization on your template DNA sequence to match the specific tRNA abundance profile of the extract’s origin organism.

    4. Reason: The protein is generated in high quantities but is also utilized very quickly because of high demand from downstream pathways.
      Fix: Perform pathway analysis using Computational and Systems Biology to block downstream mechanisms allowing the target protein to accumulate…

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?

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

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

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

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

a. What would be the membrane made of?

b. What would you encapsulate inside? Enzymes, small molecules.

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 promoters, like Tet-ON, you need mammalian)

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

  1. 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.)

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

Design of a Synthetic Minimal Cell (SMC) for Environmental Contaminant Sensing

  • 1. Function and System Logic
    • Concept: A synthetic minimal cell designed as an environmental diagnostic tool to detect localized contamination of the agricultural pollutant atrazine.
    • Input/Output: The input is extracellular atrazine (which freely diffuses across the lipid membrane). The output is the molecular release of IPTG into the surrounding environment.
    • Realization without encapsulation: No, this cannot be accomplished using open cell-free TX-TL alone. Without the physical barrier of a lipid membrane, the signaling molecule (IPTG) would diffuse immediately into the media and trigger downstream reporter systems blindly, eliminating the conditional sensor gating.
    • Realization via natural cells: Yes, an atrazine-responsive riboswitch could be engineered into live bacteria. However, using an SMC eliminates the risk of genetic drift, mutation, or cellular death caused by environmental selection pressure or toxicity.
    • Desired Outcome: In the presence of atrazine, the SMC synthesizes a pore-forming protein, allowing internal IPTG to escape and activate nearby engineered E. coli biosensors, transforming them into a bright, visible fluorescent green indicator line.

  • 2. Component Architecture and Communication
    • Membrane Composition: A structural lipid bilayer composed of POPC (Palmitoyl-oleoyl-phosphatidylcholine) supplemented with cholesterol to control membrane fluidity and minimize unmediated small-molecule leakage.
    • Encapsulated Material: Crude E. coli TX-TL extract, a baseline pool of free internal IPTG molecules, and an expression plasmid encoding a pore-forming channel controlled by an upstream atrazine-binding RNA riboswitch.
    • Extract Strain Origin: A bacterial transcription/translation system (E. coli extract) is chosen because prokaryotic riboswitches and standard RNA aptamers integrate seamlessly with bacterial translation machinery.
    • Environmental Communication: The input molecule (atrazine) is small and hydrophobic enough to pass through the lipid membrane naturally. The output molecule (IPTG) is membrane-impermeable and remains trapped inside until the atrazine triggers the transcription and translation of the membrane channels.

  • 3. Experimental Details and Validation
    • Lipids and Specific Genes:
      • Lipids: POPC and Cholesterol.
      • Genes: The Alpha-hemolysin (aHL) pore gene, fused directly downstream of an engineered atrazine riboswitch aptamer loop sequence.
      • Reporter Cells: Non-pathogenic E. coli transformed with a simple GFP reporter gene driven by a standard T7 promoter and a Lac operator.
    • Functional Measurement: The SMCs and reporter bacteria are co-cultured in solution. Following exposure to varying concentrations of atrazine, the activation profile is evaluated by tracking bulk green fluorescence output over time using a P51 Molecular Fluorescence Viewer or a standard laboratory plate reader.

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.

    • An interactive smart jacket woven with freeze-dried, cell-free biosensors that changes colour when it detects airborne toxic organophosphate pesticides in agricultural fields.

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

    • The fabric of the jacket is embedded with freeze-dried E. coli extract, a constitutive transcriptional setup, and a specific butyrylcholinesterase enzyme logic circuit. When an agricultural worker is exposed to pesticide overspray, the ambient moisture or sweat rehydrates the cell-free reagents woven into the fibres. The target organophosphates immediately inhibit the enzyme circuit, stopping a background reaction and causing a visible, safe enzymatic colour change across the sleeve.

  • What societal challenge or market need will this address?

    • It addresses acute chemical exposure and long-term poisoning risks faced daily by agricultural labourers worldwide, providing an immediate, low-cost wearable warning system without requiring complex electronic displays.

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

    • The cell-free system is stabilized inside porous paper matrices stitched as modular, disposable patches into the jacket’s shoulders and sleeves. It remains completely inert while dry, activates instantly upon absorbing atmospheric droplets or fluid, and can be cleanly unclipped and replaced with a fresh dry patch after a positive exposure event.

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 BioBitsÂŽ cell-free protein expression system, but you may also use the other tools in the Genes in Space toolkit (the miniPCRÂŽ thermal cycler and the P51 Molecular Fluorescence Viewer). For more inspiration, check out https://www.genesinspace.org/.

  • 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)

    • Long-duration space travel exposes astronauts to chronic cosmic radiation, driving high rates of DNA double-strand breaks and cellular oxidative stress. Monitoring this progressive molecular damage in real time is a critical challenge for crew safety. Current medical diagnostic platforms require heavy, power-hungry equipment that is infeasible for long missions. Developing zero-footprint, lightweight biological monitoring solutions is essential for tracking astronaut health and testing the effectiveness of spacecraft radiation shielding materials over time.

  • 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)

    • The target is the expression of the human p53 protein, a universal cellular biomarker that activates exponentially in response to direct DNA damage and radiation stress.

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

    • The p53 pathway serves as the primary master regulator for DNA damage responses in human cells. By monitoring the real-time transcription and translation of this specific molecular target under microgravity conditions, we can directly gauge the precise kinetic rate and cellular intensity of radiation-induced mutations. This provides an immediate, quantifiable readout of the current onboard radiological threat level to human tissue.

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

    • Hypothesis: A freeze-dried cell-free expression system can be successfully rehydrated aboard the International Space Station (ISS) to accurately quantify radiation damage in blood samples without active cell culture maintenance. Because cell-free systems bypass the metabolic requirements of living organisms, they remain structurally stable during long-term storage in microgravity, providing a reliable, low-resource diagnostic platform.

  • 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)

    • Astronaut blood samples will be collected, and their isolated DNA will be mixed with BioBitsÂŽ freeze-dried extracts containing a customised p53-activated fluorescent aptamer plasmid. Samples will be incubated inside the miniPCRÂŽ thermal cycler, and visual output will be captured using the P51 Molecular Fluorescence Viewer. Control reactions will include a pre-flight radiation-damaged positive control and a shielded ground-control setup to isolate space-specific baseline drift.

Homework Part B: Individual Final Project

I submitted my Twist order late, but then realised a few changes were needed! I request that a few twist orders be allowed to process in the next semester for Global Committed Listeners. This will help us a lot as we have refined our individual projects together…