Week 9 HW: Cell Free Systems

Cell-Free Protein Synthesis

Question 1 — Advantages of Cell-Free Protein Synthesis Over Traditional In Vivo Methods

Cell-free protein synthesis (CFPS) offers several key advantages over conventional cell-based (in vivo) expression systems, primarily in terms of flexibility and experimental control.

Flexibility

Unlike living cells, cell-free systems are open reaction platforms. Researchers can directly manipulate the reaction environment by adding or withholding any component — cofactors, chaperones, non-natural amino acids, labeling agents, or inhibitors — at any point during the reaction. There are no cell membranes limiting access to the transcription/translation machinery, and no cellular growth constraints.

Control Over Experimental Variables

CFPS allows fine-tuned control over:

• Concentration of DNA template (linear or circular)
• Redox potential (relevant for disulfide bond formation)
• Temperature, pH, and ionic strength
• Protease and nuclease activity (through inhibitor supplementation)
• Stoichiometry of translation factors and chaperones

This level of control is virtually impossible in a living cell without massive genetic engineering.

Two Cases Where Cell-Free Expression Is More Beneficial

Question 2 — Main Components of a Cell-Free Expression System

A typical cell-free expression system contains the following core components:

1. Cell Extract (Cytoplasmic Lysate)

The biological “engine” of the system. Prepared by lysing cells (commonly E. coli, wheat germ, rabbit reticulocytes, insect cells, or CHO cells) and removing cell debris and genomic DNA by centrifugation. It provides:

• Ribosomes and ribosomal subunits
• Translation initiation, elongation, and termination factors
• Aminoacyl-tRNA synthetases
• Molecular chaperones
• RNA polymerases (if prokaryotic)

2. DNA or mRNA Template

Provides the genetic instructions for the target protein. Can be supplied as:

• Plasmid DNA (requires transcription by RNA polymerase)
• Linear PCR product (fast and flexible, no cloning required)
• Pre-synthesized mRNA (bypasses transcription, useful in eukaryotic systems)

3. Amino Acids

All 20 standard amino acids (plus any desired ncAAs) must be supplied in sufficient concentrations as building blocks for the polypeptide chain.

4. Energy Regeneration System

Provides and replenishes ATP and GTP, which are consumed rapidly during translation. Common solutions include phosphocreatine/creatine kinase, phosphoenolpyruvate (PEP)/pyruvate kinase, or glucose-6-phosphate systems (see Q3).

5. NTPs (Nucleoside Triphosphates)

ATP, GTP, CTP, and UTP are required for RNA synthesis (transcription) and ribosome function. ATP and GTP are particularly critical.

6. Salts and Buffer System

A buffered solution (often HEPES or Tris) at physiological pH (~7.5), with optimized concentrations of Mg²⁺ (crucial for ribosome function), K⁺, and other ions.

7. Cofactors and Supplementary Additives

Depending on the application, these may include:

• Spermidine and putrescine (stabilize ribosomes)
• DTT or glutathione (control redox for disulfide bonds)
• Protease inhibitors (prevent target protein degradation)
• Chaperones (assist proper folding of complex proteins)

Question 3 — Why Energy Regeneration Is Critical and How to Ensure Continuous ATP Supply

Why It Is Critical

Protein synthesis is among the most energetically expensive cellular processes. Each peptide bond formation consumes at least 4 ATP equivalents (2 ATP for aminoacyl-tRNA charging, 1 GTP for elongation factor Tu, 1 GTP for translocation by EF-G). Additionally, transcription, mRNA capping, and molecular chaperone activity all consume ATP/GTP. Since cell-free systems contain a finite pool of nucleotides and no mitochondria or oxidative phosphorylation, ATP is depleted within minutes without an exogenous regeneration system — halting protein synthesis entirely.

Methods for Continuous ATP Supply

The most widely used approach is the phosphocreatine / creatine kinase (PCr/CK) system:

Phosphocreatine + ADP → Creatine + ATP (catalyzed by creatine kinase)

Experimental implementation:

1. Add 20–80 mM phosphocreatine directly to the cell-free reaction.
2. Supplement with purified creatine kinase (CK) (~0.1–1 mg/mL).
3. CK continuously regenerates ATP from ADP as it is consumed by ribosomes and other ATPases, extending the productive reaction time.
4. Monitor reaction progress and, for long-duration experiments, use a fed-batch or dialysis-based system to replenish PCr and remove inhibitory inorganic phosphate (Pᵢ) that accumulates over time.

Alternative systems include phosphoenolpyruvate (PEP)/pyruvate kinase, glucose-6-phosphate, or the more advanced oxidative phosphorylation-coupled systems using maltose/glucose as substrates for sustained multi-hour synthesis.

Question 4 — Prokaryotic vs. Eukaryotic Cell-Free Expression Systems

Chosen Proteins and Justification

Prokaryotic system — Choice: T7 RNA Polymerase

T7 RNA polymerase is a relatively small (~99 kDa), single-subunit bacterial enzyme with no requirement for glycosylation or complex eukaryotic PTMs. E. coli-based CFPS yields are typically high for such soluble bacterial proteins. It can be produced rapidly in a batch E. coli lysate system and used directly in downstream cell-free reactions — making prokaryotic CFPS an efficient, cost-effective choice.

Eukaryotic system — Choice: Erythropoietin (EPO)

EPO is a 34 kDa glycoprotein hormone where glycosylation accounts for ~40% of its molecular weight and is essential for its in vivo half-life, solubility, and biological activity. Prokaryotic systems cannot perform N- and O-linked glycosylation. A CHO-based or insect cell-based CFPS system provides the necessary glycosylation machinery, disulfide bond isomerases (for its two disulfide bridges), and signal peptide processing — making eukaryotic CFPS the only rational choice for functionally relevant EPO production.

Question 5 — Designing a Cell-Free Experiment for Membrane Protein Expression

Challenges of Membrane Protein Expression in CFPS

Membrane proteins (MPs) represent >30% of all encoded proteins but are notoriously difficult to produce because:

1. Hydrophobic transmembrane (TM) domains cause aggregation and precipitation in aqueous cell-free reactions.
2. Lack of a lipid bilayer means TM segments have no natural environment to insert into.
3. Correct topology and oligomeric state are difficult to achieve without a membrane.
4. MPs are prone to misfolding and protease degradation.

Experimental Design

Step 1 — Template Preparation

• Clone the MP gene into a T7-promoter vector.
• Include an N-terminal His-tag or Strep-tag for downstream detection and purification.
• Optionally, codon-optimize for the expression host (E. coli lysate is common for MPs).

Step 2 — Choose a Solubilization Strategy (Critical Decision)

Three main strategies exist:

Recommended approach: Start with detergent screening (DDM at 0.1–1% w/v) combined with lipid nanodisc supplementation for functional studies.

Step 3 — Reaction Optimization

• Use a batch or dialysis mode reaction at 30°C (reduces aggregation vs. 37°C).
• Supplement with lipids (DOPG, DOPC) matching the natural membrane composition.
• Add oxidizing glutathione buffer (if the MP has extracellular disulfide bonds).
• Include chaperones (SecB, SRP analog) for co-translational support.

Step 4 — Quality Control and Detection

• Run an SDS-PAGE + western blot using the affinity tag to verify expression.
• Test solubility by centrifugation (100,000 × g) — soluble fraction indicates successful solubilization.
• Use fluorescence-based functional assays (e.g., ligand binding, ion flux) to confirm correct folding.

Step 5 — Iterative Optimization

Screen a matrix of:

• Detergent type and concentration
• Lipid:protein ratio
• Mg²⁺ and K⁺ concentrations
• Template DNA concentration

Question 6 — Troubleshooting Low Protein Yield in a Cell-Free System

Reason 1: Rapid ATP/Energy Depletion

Mechanism: Without adequate energy regeneration, translation halts prematurely. This is the most common cause of low yield.

Troubleshooting strategy:

• Measure inorganic phosphate (Pᵢ) accumulation over time using a colorimetric assay — high Pᵢ indicates energy system exhaustion.
• Switch from a batch mode to a dialysis (CECF — continuous exchange cell-free) mode, where fresh energy substrates are continuously supplied through a dialysis membrane while inhibitory byproducts (Pᵢ, PPᵢ) are removed.
• Optimize the concentration of phosphocreatine (try 20, 40, 60, 80 mM) and verify CK activity.

Reason 2: mRNA Instability or Insufficient Transcription

Mechanism: Cell extracts contain ribonucleases (RNases) that degrade mRNA rapidly. If mRNA is short-lived, ribosomes have no template to translate, drastically reducing yield.

Troubleshooting strategy:

• Add RNase inhibitors (e.g., RiboLock, SUPERase-In) to the reaction at the start.
• Check mRNA levels at time points (0, 30, 60 min) by extracting RNA and running an agarose gel or RT-qPCR.
• Optimize the T7 RNA polymerase concentration if using a coupled transcription-translation system.
• Use a circular plasmid instead of a linear PCR product (linear DNA is more susceptible to exonuclease degradation unless protected with phosphorothioate end caps).
• Ensure the 5’UTR contains a strong ribosome binding site (RBS) such as the Shine-Dalgarno sequence (prokaryotic) or IRES element (eukaryotic).

Reason 3: Target Protein Degradation or Aggregation Post-Synthesis

Mechanism: Even if the protein is synthesized in adequate amounts, it may (a) misfold and aggregate into insoluble inclusion body-like structures within the reaction, or (b) be degraded by proteases remaining in the extract.

Troubleshooting strategy:

• Distinguish aggregation from degradation: Centrifuge the reaction at 10,000 × g and run both pellet (insoluble) and supernatant (soluble) fractions on western blot. If most protein is in the pellet → aggregation; if total protein is low in both fractions → degradation.
• For aggregation: Add molecular chaperones (DnaK/DnaJ/GrpE, GroEL/GroES) exogenously; lower reaction temperature to 25–30°C; reduce DNA template concentration (slower synthesis rate allows more time for folding).
• For degradation: Add a protease inhibitor cocktail (PMSF, leupeptin, pepstatin A) at the start of the reaction; use a protease-deficient extract prepared from ΔompT Δlon E. coli strains.

Answers compiled from core principles of cell-free expression biology. Key references: Gregorio Georgiou & Lydia Kirsanova CFPS reviews; Pardee et al. (2016) Cell; Silverman et al. (2019) Nature Protocols.

Homework question from Kate Adamala

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

1. Pick a function and describe it.

• What would your synthetic cell do? What is the input and what is the output?
• Would this function be realized by cell-free Tx/Tl alone, without encapsulation?
• Could this function be realized by genetically modified natural cell?
• Describe the desired outcome of your synthetic cell operation.

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

• What would be the membrane made of?
• What would you encapsulate inside? Enzymes, small molecules.
• 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)
• How will your synthetic cell communicate with the environment? (hint: are substrates permeable? or do you need to express the membrane channel?)

3. Experimental details

• List all lipids and genes. (bonus: find the specific genes; for example, instead of just saying “small molecule membrane channel” pick the actual gene.)
• How will you measure the function of your system?

Homework question from Peter Nguyen

1. 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.
• How will the idea work, in more detail? Write 3-4 sentences or more.
• What societal challenge or market need will this address?
• How do you envision addressing the limitation of cell-free reactions (e.g., activation with water, stability, one-time use)?

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)
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)
3. Describe how your molecular or genetic target relates to the space biology question or challenge your proposal addresses. (Maximum 100 words)
4. Clearly state your hypothesis or research goal and explain the reasoning behind it. (Maximum 150 words)
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)