Week 9: Cell-Free Systems

Part A: General and Lecturer-Specific 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.

In a cell-free system, the reaction environment is fully open and directly accessible. The researcher can manipulate temperature, pH, ionic strength, redox potential, and cofactor concentrations in real time. This is not possible in a living cell without perturbing global physiology. The DNA template is added exogenously, meaning you can switch between constructs instantly without cloning into an expression vector, transforming cells, and growing overnight cultures. Toxic or unnatural amino acids can be incorporated freely because there is no selective pressure to keep the cell alive. Reaction volumes are scalable from a few microlitres to litres without the complexity of fermenter optimization.

There are cases where cell-free systems are superior. Many proteins, e.g. pore-forming toxins, viral proteins, potent enzymes, etc. kill the host cell before useful quantities accumulate. In a cell-free system there is no cell to kill, so expression proceeds unimpeded. A classic example is the synthesis of bacteriophage lysis proteins or cytotoxic anticancer peptides. Another example is the overexpression of integral membrane proteins in living cells which saturate the insertion machinery, cause membrane stress, and is usually lethal or leads to inclusion body formation. Cell-free systems allow the co-addition of detergents, liposomes, nanodiscs, or styrene–maleic acid lipid particles (SMALPs) directly into the reaction, enabling co-translational solubilization and folding in a controlled lipid environment.

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

• Lysate: Supplies ribosomes, translation factors (initiation, elongation, release), chaperones, tRNA synthetases, and all endogenous enzymes needed for transcription/translation.

• DNA template: Carries the gene of interest under a suitable promoter (T7, SP6, or σ70); plasmid, PCR product, or linear DNA are all viable.

• RNA polymerase: Transcribes the DNA into mRNA; T7 RNAP is most commonly added exogenously for high-yield prokaryotic systems.

• Amino acids: Substrates for peptide-bond formation; all 20 canonical amino acids (or non-canonical analogues) must be supplied.

• Energy regeneration system: Provides and recycles ATP/GTP to power translation; typically creatine phosphate/creatine kinase or phosphoenolpyruvate/pyruvate kinase.

• Salts and buffer: Mg²⁺ and K⁺ concentrations are critical for ribosome activity; HEPES or TRIS maintains pH stability.

• tRNAs: Charged by aminoacyl-tRNA synthetases in the extract; occasionally supplemented for non-canonical amino acid incorporation.

• Cofactors and additives: Spermidine, putrescine stabilise ribosomes; DTT or glutathione controls redox potential for disulfide-bond formation.

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.

Translation is energetically expensive since each peptide bond consumes at least two ATP equivalents, and tRNA charging, GTP hydrolysis by elongation factors, and mRNA synthesis add further demand. The small volume of a cell-free reaction exhausts its initial ATP pool within minutes. Once ATP falls below ~0.5 mM, translation stalls irreversibly, so continuous regeneration is essential for practical yields.

The most widely used approach in E. coli-based CFPS is the creatine phosphate/creatine kinase system. Creatine kinase catalyzes:

Creatine phosphate + ADP → Creatine + ATP

You add creatine phosphate (typically 20–80 mM) as the phosphate donor and creatine kinase (80–200 µg/mL) as the enzyme to the reaction mix. As ATP is consumed by ribosomes, the equilibrium is continuously driven toward ATP regeneration. The reaction sustains translation for 1–6 hours depending on system quality.

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

T7 RNAP is a large, single-subunit bacterial enzyme (99 kDa) that does not require glycosylation or eukaryotic chaperones for folding. It is produced at high yield, is robustly active when expressed in E. coli CFPS, and is itself used as a component of prokaryotic cell-free systems making E. coli CFPS the natural and cost-efficient choice. The high yield and speed of the prokaryotic system allow rapid iterative mutagenesis studies of polymerase variants.

EPO is a 30.4 kDa human glycoprotein hormone where three N-linked and one O-linked glycan chains are essential for biological activity, serum half-life, and receptor binding. These modifications cannot be added by prokaryotic ribosomes. A CHO cell-free or insect-cell-free system provides the glycosyltransferases, oligosaccharyltransferase complexes, and the endoplasmic reticulum membrane environment needed to glycosylate EPO co-translationally, making it the only appropriate cell-free platform for producing biologically active EPO.

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 are inherently hydrophobic. Without a lipid bilayer, the transmembrane helices aggregate, misfold, or precipitate. In whole-cell expression, this problem is partially mitigated by the membrane insertion machinery (SecYEG translocon in prokaryotes, Sec61 in eukaryotes), which is absent in a standard lysate. Cell-free systems must therefore provide an artificial hydrophobic environment co-translationally.

Use a plasmid or linear PCR product with a T7 promoter driving the gene, with the native signal anchor or TM helices intact. Avoid N-terminal his-tag placement if it sterically interferes with membrane insertion; prefer a C-terminal tag or cleavable N-terminal tag.

Three main strategies exist and should be tested in parallel:

1. Add digitonin, DDM (n-dodecyl-β-D-maltoside), or LMNG at concentrations near but below CMC during translation. The nascent hydrophobic segments partition into detergent micelles rather than aggregating.
2. Pre-form unilamellar liposomes from a defined lipid mixture (e.g., DOPC:DOPE:cholesterol mimicking the ER membrane). Adding them during translation allows co-translational insertion.
3. Pre-assembled nanodiscs (MSP protein + defined lipids) or SMALPs provide a discoidal bilayer patch. The protein inserts co-translationally, remaining surrounded by a native-like bilayer.

Many membrane proteins contain extracellular disulfide bonds. Adjust the glutathione ratio (oxidised:reduced = 4:1 to 1:1) to create a mildly oxidising environment that supports disulfide formation without inhibiting the translation machinery. Add purified bacterial or eukaryotic chaperones (e.g., GroEL/GroES for bacterial targets; Hsp70/Hsp40/Hsp90 for eukaryotic channels) to assist folding of soluble domains connected to the TM segments. For a eukaryotic receptor (GPCR, ion channel), prefer a wheat germ or insect-cell lysate for its ability to support signal peptide cleavage and glycosylation. For a bacterial transporter, an E. coli lysate is appropriate. Assess yield by fluorescence (GFP fusion or FSEC — fluorescence-size exclusion chromatography), check functionality by ligand binding or electrophysiology in reconstituted liposomes, and confirm topology by limited proteolysis or cysteine-accessibility assays.

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: Rapid mRNA Degradation Cell lysates contain endogenous RNases that degrade the transcript before sufficient translation occurs, particularly for linear DNA templates or mRNAs with AU-rich 3′ sequences.

First, assess mRNA stability by adding a transcription inhibitor (rifampicin) after a brief transcription period and quantifying mRNA by RT-qPCR at time points. To remedy degradation: (a) use a closed circular plasmid rather than linear DNA, which is more nuclease-resistant; (b) add RNase inhibitor (e.g., RiboLock or SUPERase•In) to the reaction; (c) incorporate a 5′ stem-loop structure (e.g., from bacteriophage ϕ10 leader) or a 3′ poly-A tail to protect mRNA termini; (d) optimise the 5′ UTR to include an efficient ribosome-binding site (Shine–Dalgarno in prokaryotic systems).

• Reason 2: Suboptimal Mg²⁺ or K⁺ Concentration Ribosome assembly, aminoacyl-tRNA binding, and many GTPase activities are acutely sensitive to Mg²⁺ concentration. The optimal free Mg²⁺ for E. coli-based CFPS is typically 6–12 mM but varies between lysate batches because endogenous metabolites chelate magnesium unpredictably.

Perform a two-dimensional titration of Mg²⁺ (4–18 mM in 2 mM steps) and K⁺ (50–250 mM in 50 mM steps) in small-volume (10–15 µL) reactions with a reporter protein (e.g., GFP or luciferase) before switching to your target. Identify the peak of reporter yield; this optimal ionic condition is then transferred to your target protein reactions. This is one of the most impactful optimisations in any new CFPS setup.

• Reason 3: Protein Insolubility / Aggregation The target protein may be translated efficiently but immediately misfolds and precipitates, rendering it undetectable in the soluble fraction by standard western blot or ELISA.

Centrifuge the reaction at 15,000 × g for 10 min and separately analyse the supernatant and pellet fractions. If the target is enriched in the pellet, aggregation is the issue. Remedies include: (a) lowering the reaction temperature from 37°C to 25–30°C to slow translation and give chaperones time to act; (b) supplementing with purified chaperones (GroEL/GroES, DnaK/DnaJ/GrpE); (c) adding co-solubilising agents such as arginine (100–500 mM) or non-ionic detergents at sub-CMC concentrations; (d) fusing a solubility tag (SUMO, MBP, GB1) to the N-terminus to nucleate correct folding; and (e) for disulfide-containing proteins, systematically varying the oxidised/reduced glutathione ratio to find the redox optimum.

Homework question from Kate Adamala

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.

2. 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 promotors, 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?)

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.)

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

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.
• 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)

Part B: Individual Final Project