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.

• If we wanted to produce a toxic protein / protein deposits which are misfolded, non-functional, and often highly stable multi-molecular structures - restriction enzymes which cut and edit DNA, cytotoxic proteins (e.g. immune cell related molecules such as perforins which puncture cell membranes and granzymes which trigger cell apoptosis, both produced by NK cells), the cell free expression allows production without affecting cell viability in comparison to in vivo methods.
• Due to the barrier of cell membranes being removed in cell-free systems, labeled or unnatural amino acids can be put in the mix (of organelles like ribosomes) for targeted, specialised protein synthesis: for example in in vivo labeling for NMR or fluorescence studies.
• In in vivo methods, the cell system’s energy and resources can be divided between different organelles, whereas in CFS the energy and resources can solely be focused on making the target protein.
• You can do real time tracking of protein synthesis using spectrophotometry or other analytical methods in CFS due to the large, uncomplicated system comparatively to in vivo methods where the cell has many organelles.
• Conditions of the reaction can be controlled; variables such as temperature, redox potential and pH can be changed and finetuned without worrying about causing host death.
• Cloning and transformation steps are skipped, as we can use linear DNA or circular plasmid DNA directly.

Two cases where CFS is beneficial over in vivo methods;

1. We will save time and resources by skipping cloning and transformation expected in in vivo systems as we have the ability to synthesise from directly from linear PCR products. This could be for example if we wanted to screen many thousands of protein mutants for functional genomics / discovery for drugs reasons.
2. Addition of non-natural amino acids without worrying about travelling across the cell membrane present in in vivo systems. We can use this for area specific labelling with fluorescent dyes or stable isotopes for NMR and X-ray crystallography.

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

Components include;

1. Cell machinery - ribosomes for mRNA to protein translation, enzymes (Aminoacyl-tRNA Synthetases, RNA polymerase), translation factors
2. Circular plasmids / linear DNA - genetic material for replication
3. Nucleoside triphosphates - energy generation for cell processes
4. Small molecules and buffer - amino acids, building blocks of proteins, Mg2+ and K ions for stability of ribosome as cofactors and enzyme active site driven catalytic activity.
5. Buffers to maintain stable pH
6. Agents that prevent oxidation during protein synthesis

[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.

Creatine Phosphate (CP) / Creatine Kinase (CK enzyme) system can ensure a continuous ATP supply in a CFS, producing creatinine which is a byproduct which does not interfere with cell translation machinery, as usually in the CFS, ATP hydrolysis produces increasing levels of inorganic phosphate which inhibit protein synthesis by sequestering essential magnesium ions (Whitaker, 2013).

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

GFP would be suitable to produce in a prokaryotic system as it does not require PTMs and is a non glycolysated protein. It is a non complex protein and does not require complicated conformational folding into a 3D functional structure. As E. coli systems can do protein synthesis quickly in terms of speed and volume, they are suitable for high-throughput screening of fluorescent reporter proteins like GFP, in order to verify system activity / test varying promoter strengths without the energy and resources used in eukaryotic processing.

Human Tissue Plasminogen Activator (tPA) would be suitable to produce in a eukaryotic system as it requires PTM (multiple disulfide bonds and specific glycosylation patterns to become a functional protein). Since it is a complex protein, if produced in the prokaryotic system it would make an inactive, misfolded inclusion body, which can be toxic and nonfunctional, of tPA. A eukaryotic system (Chinese hamster ovary / insect cell-free) provides the protein chaperones and microsomal membranes needed for correct protein folding and will receive the sugar chains required for its function, thrombolysis in blood clotting.

[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.

1. Select expression platform - eukaryotic (PTMs, microsomes can assist in protein folding for complex proteins) or prokaryotic (no PTMs, but fast and large amount of proteins produced)
2. Create a synthetic phospholipid bilayer - use liposomes/vesicles directly to the reaction to provide a landing site for the hydrophobic transmembrane regions and use membrane scaffold proteins (MSPs) to produce nanodiscs which catch the protein in its native state and use detergents for protein solubility if lipids not used
3. Sequence and Template Optimization for ribosome mRNA reading and translation - smoothes out mRNA knots at the N-terminus and N-terminal tags as ribosome handles.

Challenges and solutions

Protein misfolding which produces a nonfunctional 3D structure –> produce the protein whilst there are nanodiscs / liposomes to provide immediate hydrophobic shielding. Low yield –> Implement a continuous exchange (CECF) system. We’ll use a slightly permeable lipid membrane to give them fresh nutrients and remove inhibitory byproducts, increase the reaction time. Incomplete folding –> add molecular chaperones (like DnaK/DnaJ) or utilise eukaryotic cell lysates which contain protein folding chaperones in microsomes. Batch variability –> Utilise AI-driven active learning to standardise reaction protocols across various lysate batches.

[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.

• Incomplete template or codon bias: Incorrect DNA sequences or rare codons can stall translation, we can troubleshoot by verifying the template integrity via sequencing or using codon-optimised genes.

• Resource depletion or inhibitors: High levels of metabolic byproducts or nucleases can degrade components; troubleshoot by using a continuous-exchange system or adding RNase inhibitors and fresh energy substrates.

• Protein folding or solubility issues: Rapid synthesis can lead to misfolded proteins / inclusion bodies; we can troubleshoot by adding molecular chaperones or reducing the reaction temperature to slow down the translation rate.

Homework question from Kate Adamala Based on: Tang et al., 2017

Pick a function and describe it.

What it does: The synthetic cell acts as a translator which converts an enzyme-based signal into a genetic output. Input: Glucose and an enzyme (Glucose oxidase which oxidises glucose into gluconic acid and hydrogen peroxide, and is often used in blood glucose level tracking, extract oxygen from bottled drinks and food packaging (such as mayonnaise, wine) to increase its shelf life by preventing oxidation and bacterial proliferation. Output: Hydrogen peroxide which triggers a genetic response (protein expression) in a neighboring population, GOx-produced Hydrogen peroxide can alter genetic expression by inducing oxidative stress-mediated pathways, activating stress-response mechanisms, and triggering cell-specific defence pathways like for e.g. p53, and repressing anti-aging genes (like Klotho).

Could this function be realized by cell-free Tx/Tl alone?

No, without encapsulation, the chemical gradients needed to trigger specific downstream signalling would dissipate. The protocell environment provides a high local concentration of DNA and transcription machinery, which would be too diluted in an open solution.

Could this function be realized by genetically modified natural cell?

Methodically yes, but natural cells have a composite metabolism that might interfere with the specific chemical intermediates (Hydrogen Peroxide). A synthetic cell allows for a noise-free channel: removing background noise from the cell’s own redox homeostasis.

What would the membrane be made of?

This paper uses protein-polymer microcapsules (proteinosomes), for my assignment, I’d want to adapt it to POPC/POPG phospholipids to create vesicles that mirror natural anionic bacterial membranes more closely than single-lipid systems.

What would you encapsulate inside?

• Bacterial Tx/Tl machinery (like PURE system).

• Plasmids encoding the target protein.

• Small molecule substrates (e.g., specific nucleotides and amino acids).

Which organism will your Tx/Tl system come from?

• E. coli species - bacterial strain. This is because the promoters used in these chemical communication studies (like the oxyR system for Hydrogen peroxide sensing) are natively bacterial.

How will your synthetic cell communicate? The membrane is designed to be semi-permeable to small molecules (glucose, Hydrogen peroxide) but impermeable to the large DNA and enzymes inside. I may use Alpha-hemolysin (aHL) pores to ensure the exit of larger output molecules.

Experimental details (Genes/Lipids)

Lipids: POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine).

Genes: The oxyS promoter (which is induced by oxidative stress) controlling the expression of mCherry or GFP.

Pore Gene: hla (encodes Alpha-hemolysin).

How will you measure function? I’ll use Fluorescence Microscopy to see single synthetic cells glow after receiving the chemical signal.

I’ll use a Plate Reader for measuring the kinetics of the protein expression over time across the whole population.

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.

My proposal is using FDCF systems embedded within a 3D-printed, biocompatible soft robotic lattice. The system will be a bridge between a robotic prosthetic and human tissue, capable of sensing physiological states (such as neuroendocrine activation, high energy use, insulin resistance, inflammatory and immune system response, disregulated electrolyte and fluid levels, increased blood clotting and gastrointestinal issues such as vomiting and nausea) and monitoring post-surgical tissue health in real-time and responding autonomously by secreting therapeutic enzymes to prevent infection and increase tissue healing.

How will the idea work, in more detail? Write 3-4 sentences or more. I will use FDCF genetic circuits, inside hydrogel microbeads within a 3D printed matrix and has a long shelf life until it is hydrated by ISF or sweat. It can recognise pathogen associated molecular patterns (specific antigens or epitopes that specific pathogen strains have), or inflammatory biomarkers (drop in pH / detecting specific bacterial RNA). As the Cell free system activates, it will trigger the release of antimicrobial peptides to reduce inflammation and signalling molecules which promote proliferation, differentiation, and tissue repair to increase tissue healing. The material structural properties change (e.g. swelling in a specific area of the body) to provide a mechanical signal to the user’s robotic prosthetic.

What societal challenge or market need will this address?

Sensors require batteries and wiring to produce an output; the biosthetic graft is power-independent and biologically powered. It also has the benefit of localised synthesis of antimicrobial peptides for instance for reducing bacteria related inflammatory biomarkers.

Vascular disease and diabetes patients (10%) may rely on prosthetics due to developing open sores or wounds, on the bottom of the foot, leading to complex conditions such as gangrene which can lead to limb amputation. Patients with neuropathy cannot feel when a prosthetic causes a pressure related sore or if a wound turns into a bacterial infection. Due to this being noticed late, the patient’s symptoms can be irreversibly damaging. One size of prosthetic does not fit everyone, as limbs change size and shape during the day because of swelling or temperature. A static prosthetic cannot adapt, leading to friction between the skin and prosthetic and skin breakdown. Therefore the FDCF solution I proposed finds the infection before symptoms occur, and is a lattice featuring macro-pores - large allow the skin to breathe and preventing skin maceration, micropores which wick the interstitial fluid and deliver to the embedded cell-free sensors and nanopores which allows sugars and RNA to go to the gene circuits, for gradient stiffness: a 3D printed graft which is viscoelastic. It will have regenerative properties too, if a small tear appears in the material the cell-free material can produce collagen resembling peptides to be self healing and repair the microtear. We can use fluorescent repair proteins like GFP to report levels of bacterial infection when they are high and the graft can change colour to represent this to the patient, alongside a light output warning them.

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

Regarding one time use and constant topping of water for activation of the FDCF via microfluidic gating, where the robot’s CPU accurately hydrates specific FDCF pixels when required, and using trehalose and silica (pairing of a protective sugar and a mineral material) to lock components in a long shelf life, glassy state. This would allow multiple uses of the same graft.

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/ .

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)

The question I want to address: Can you engineer yeast or bacteria to produce medicine on demand in space?

Many Earth-made medicines can expire or degrade quickly due to space radiation. I want to Program genetic circuits that trigger the production of vitamins or antibiotics only when needed. This is relevant, as it will reduce the massive cargo weight of medical supplies for deep-space transit.

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) My target will be PCUPI and PGAL1, inducible promoters which will initiate drug synthesis in the presence of a non-lethal copper source and galactose being added respectively.

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

My target will be the inducible promoters, Copper-inducible promoter (PCUPI) and galactose-inducible promoter (PGAL1). The promoters are tunable and inducible meaning certain conditions such as the presence of ions are needed for an output (in this case drug synthesis). PCUPI allows astronauts to start drug synthesis primarily when needed by adding a non-lethal copper source, reducing the metabolic burden on the yeast in contrast to constitutive promoters. Yeast deletion collections have shown that yeast can withstand space radiation and microgravity, making it a reliable chassis for genetic engineering in space. This has been proven in space, it been successfully used to express human proteins (like gelatin) in related yeasts and is a reliable, high-yield system. Studies have shown that is active in Pichia pastoris with copper induction within 2 hours, making it highly suitable for rapid-response drug production.

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

The goal is to develop an on-demand bioproduction system in Saccharomyces cerevisiae using a dual-input genetic switch to ensure metabolic efficiency in deep space. I hypothesise that a hybrid promoter system will allow for tighter, tunable control of medicinal protein synthesis. In this model, galactose acts as the primary “on” switch, while copper ions provide a secondary rheostat to modulate expression levels. This prevents the “leaky” expression often seen in single-inducer systems, which can lead to metabolic burden or plasmid instability during long-term storage in microgravity. By decoupling growth from production, we can maintain healthy “starter” cultures and trigger high-yield medicine synthesis only when specifically required.

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)

This experiment uses the BioBits® system to validate a hybrid promoter for on-demand protein synthesis. We will test three DNA templates: a wild-type (negative control), a constitutive promoter (positive control), and an experimental hybrid promoter driving GFP. To initiate synthesis, we will add varying concentrations of Galactose (0–2%) and Copper Sulphate (0–500 µM) to the BioBits pellets. We will use the miniPCR® for precise incubation at 37°C. Data collection includes real-time fluorescence quantification using the P51 Viewer to determine the optimal inducer ratio and maximum protein yield in microgravity.

References

Tang, T.-Y. D., Cecchi, D., Fracasso, G., Accardi, D., Coutable-Pennarun, A., Mansy, S. S., Perriman, A. W., Anderson, J. L. R., & Mann, S. (2018). Gene-mediated chemical communication in synthetic protocell communities. ACS Synthetic Biology, 7(2), 339–346. https://doi.org/10.1021/acssynbio.7b00306

Whittaker, J. W. (2013). Cell-free protein synthesis: The state of the art. Biotechnology Letters, 35(2), 143–152. https://doi.org/10.1007/s10529-012-1075-4