Lab (Week 9) — Cell-Free Systems
Completion status:
- This lab was completed theoretically (no physical or virtual wet lab performed).
- All procedures, results, and analyses below are based on the provided protocol and scientific literature.
- The homework questions are answered in full.
Overview & Objective
In this lab, we demonstrate the functionality of a Cell-Free Transcription-Translation (TXTL) system using an E. coli extract. We express the reporter protein amilGFP from a T7-IPTG‑inducible plasmid. IPTG acts as an inducer by inhibiting the LacI repressor, allowing T7 RNA polymerase to transcribe the gene. The goal is to quantify amilGFP production at different IPTG concentrations over an 8‑hour incubation at 30°C, using fluorescence measurement (ex 492 nm / em 506 nm) either in a plate reader or via end‑point imaging.
Pre‑Lab Reading Summary
1. What is Cell‑Free?
A cell‑free system uses extracted cellular components (ribosomes, RNA polymerase, tRNAs, amino acids, ATP) to carry out transcription and translation outside living cells. Advantages: no cell viability constraints, direct access to reactions, rapid prototyping.
2. TX‑TL Production
- Cell extract preparation: E. coli cells are grown, washed, disrupted (freeze‑thaw or sonication), and ultracentrifuged to obtain a lysate rich in ribosomes and enzymes. A cold chain prevents degradation.
- Master mix components (summarised in table below).
| Component | Concentration (example) | Function |
|---|---|---|
| HEPES (pH 8) | 500 mM | pH buffer |
| ATP, GTP, CTP, UTP | 15,15,9,9 mM | Nucleotides for transcription & energy |
| E. coli tRNA | 2 mg/mL | Supplies amino acids during translation |
| Folinic acid | 0.68 mM | Supports nucleotide/amino acid synthesis |
| NAD | 3.3 mM | Redox coenzyme |
| Coenzyme‑A | 2.6 mM | Acyl group transfer |
| Spermidine | 15 mM | Stabilises ribosomes/RNA |
| Sodium oxalate | 40 mM | Prevents Mg²⁺ precipitation |
| AMP | 7.5 mM | Metabolic regulation |
| 3‑PGA or PEP | 300 mM | Energy source (ATP regeneration) |
| Mg‑glutamate / K‑glutamate | variable | Cofactors for enzymes |
| DTT | variable | Reducing agent |
| T7 RNA polymerase | variable | High‑specificity transcription |
| Murine RNase inhibitor | variable | Protects mRNA from degradation |
3. PURE system vs. Whole cell extract
- PURE: defined components, lower yield, higher cost, minimal background – ideal for mechanistic studies.
- Whole cell extract: crude lysate, higher yield, cost‑effective – suitable for protein production.
Protocol (Theoretical Completion)
Materials
- E. coli AKABY cell‑free extract
- Master mix (with all components except DNA and IPTG)
- IPTG (several concentrations)
- T7-IPTG‑amilGFP plasmid (inducible GFP)
- Positive control plasmid (constitutive GFP, e.g., T7‑GFP)
- Nuclease‑free water (NFW)
- 96‑well PCR plate or PCR tubes
- Mineral oil (optional)
Day 1 – Assembly and Running
Thermocycler program (simulated):
- 30°C hold (preheat)
- 30°C for 8 hours (reaction)
- 4°C hold (stop reaction)
Reaction setup (10 µL per condition):
| Reactive | Positive control | IPTG 0.2X | IPTG 0.4X | IPTG 0.8X | Negative control |
|---|---|---|---|---|---|
| Master mix | 4.7 µL | 4.7 µL | 4.7 µL | 4.7 µL | 4.7 µL |
| Cell extract | 3.3 µL | 3.3 µL | 3.3 µL | 3.3 µL | 3.3 µL |
| IPTG (0.2X stock) | – | 1 µL | – | – | – |
| IPTG (0.4X stock) | – | – | 1 µL | – | – |
| IPTG (0.8X stock) | – | – | – | 1 µL | – |
| pDNA‑IPTG (inducible) | – | 1 µL | 1 µL | 1 µL | – |
| pDNA‑GFP (constitutive) | 1 µL | – | – | – | – |
| NFW | – | – | – | – | 2 µL |
| Total | 10 µL | 10 µL | 10 µL | 10 µL | 10 µL |
Mix gently, spin down, load into thermocycler (or plate reader). If using plate reader, add 20 µL mineral oil on top of each 10 µL reaction to prevent evaporation. Run at 30°C for 8 hours, reading fluorescence every 30 min (ex 492/em 506).
Expected result: GFP fluorescence increases over time in positive control and IPTG‑dependent samples, with higher IPTG giving faster/stronger signal up to saturation. Negative control remains near background.
Day 2 – Quantification (Simulated)
ImageJ analysis (theoretical):
- Place tubes on a blue light transilluminator, photograph.
- Open image in Fiji, select region of interest (ROI) for each tube.
- Analyze > Color Histogram – obtain mean values for red, green, blue channels.
- Because background red/blue interfere, calculate corrected green = green_mean – (red_mean + blue_mean)/2 or use ratio.
- Subtract negative control value from each sample to get net fluorescence.
- Plot net fluorescence vs. IPTG concentration.
Plate reader analysis (theoretical):
- Export kinetic traces or endpoint values (after 8 h).
- Subtract NTC (negative control) background.
- Plot fluorescence as bar graph or dose‑response curve.
Fold change calculation (example):
- Fold change = (fluorescence at IPTG X) / (fluorescence at 0 IPTG, i.e., NTC minus its own background? Actually NTC has no IPTG and no DNA, so use a “no IPTG + DNA” control if available. In the table above, the NTC has no DNA, so we cannot directly calculate fold induction from NTC. A better control would be a reaction with DNA but no IPTG (leaky expression). Since that is missing, we assume the IPTG 0.2X well already includes DNA – we compare across IPTG concentrations. So fold change relative to 0.2X: (0.4X value)/(0.2X value) etc.)
From theoretical expectation: increasing IPTG gives increasing fluorescence up to ~0.4X, then plateaus.
Homework Questions – Answered
1. Advantages of cell‑free protein synthesis over in vivo methods
Flexibility and control:
- No cell viability constraints – you can use toxic proteins, high concentrations of inducers, or non‑natural amino acids.
- Direct access to reaction – you can sample at any time, add inhibitors, or modify conditions (pH, temperature, salts) without harming cells.
- Rapid prototyping – a reaction takes hours instead of days.
Two cases where cell‑free is more beneficial:
- Toxic protein production: e.g., membrane‑active toxins or proteases that would kill host cells. In cell‑free, the protein is synthesised without affecting a living organism.
- Biosensor development: Point‑of‑care diagnostics (e.g., paper‑based freeze‑dried TXTL for detecting pathogens) – the reaction can be activated simply by adding water and sample, no need to maintain live cultures.
2. Main components of a cell‑free expression system and their roles
| Component | Role |
|---|---|
| Cell extract (lysate) | Provides ribosomes, tRNAs, aminoacyl‑tRNA synthetases, initiation/elongation factors, and often endogenous RNA polymerase (if using endogenous promoters). |
| Energy regeneration system (e.g., 3‑PGA or PEP) | Provides a continuous ATP supply via substrate‑level phosphorylation. |
| Nucleotides (ATP, GTP, CTP, UTP) | Substrates for transcription and energy (ATP). |
| Amino acids | Building blocks for protein synthesis. |
| Magnesium and potassium salts | Cofactors for ribosomes and polymerases. |
| Buffer (e.g., HEPES) | Maintains optimal pH. |
| Reducing agent (e.g., DTT) | Prevents oxidation of cysteine residues. |
| Template DNA (or mRNA) | Encodes the target protein. |
| RNA polymerase (e.g., T7) | Transcribes DNA if using a phage promoter. |
| RNase inhibitor | Protects mRNA from degradation. |
3. Why is energy regeneration critical? How to ensure continuous ATP supply?
Critical because: ATP is consumed rapidly during both transcription (NTPs) and translation (ATP for aminoacyl‑tRNA synthesis, GTP for ribosome function). Without regeneration, ATP would be depleted within minutes, stopping synthesis.
Method to ensure continuous supply: Use a secondary energy source such as 3‑phosphoglycerate (3‑PGA) or phosphoenolpyruvate (PEP) combined with the endogenous glycolytic enzymes present in the E. coli extract. 3‑PGA is converted to pyruvate via the lower glycolysis pathway, generating ATP. Alternatively, use creatine phosphate + creatine kinase. The provided master mix already contains 300 mM 3‑PGA.
4. Compare prokaryotic vs eukaryotic cell‑free systems
| Feature | Prokaryotic (e.g., E. coli) | Eukaryotic (e.g., wheat germ, rabbit reticulocyte, insect) |
|---|---|---|
| Post‑translational modifications | Minimal (no glycosylation, no disulfide bond formation efficiently) | Capable of glycosylation, phosphorylation, disulfide bonds (if supplemented) |
| Yield | High (mg/mL) | Low to medium (µg/mL) |
| Cost | Low | Higher |
| Ease of use | Simple, fast (2‑4 h) | More complex, slower (6‑24 h) |
| Protein folding | May not fold complex mammalian proteins | Better for complex, multi‑domain eukaryotic proteins |
Protein choice example:
- Prokaryotic system: Produce E. coli β‑galactosidase (LacZ) – a simple, well‑folding bacterial enzyme that needs no modifications. High yield desired.
- Eukaryotic system: Produce human erythropoietin (EPO) – requires correct disulfide bonds and N‑linked glycosylation for activity. Use wheat germ or CHO lysate supplemented with microsomes.
5. Design a cell‑free experiment to optimise membrane protein expression
Challenges:
- Membrane proteins are hydrophobic, tend to aggregate in aqueous solution.
- They require proper insertion into a lipid bilayer for folding and stability.
- Detergents or lipids can inhibit the TXTL reaction.
Design:
- Use an E. coli cell‑free system supplemented with liposomes or nanodiscs (pre‑formed lipid bilayers) to allow co‑translational insertion.
- Test different detergents at sub‑inhibitory concentrations (e.g., 0.05–0.5% digitonin, DDM).
- Use a green fluorescent protein (GFP) fusion at the C‑terminus to monitor folding and yield.
- Vary temperature (20–30°C) – lower temperature may slow synthesis but improve folding.
- Optimise magnesium and potassium concentrations (membrane protein synthesis may require higher Mg²⁺).
- Add chaperones (e.g., trigger factor, DnaK/DnaJ/GrpE) to the extract or supplement externally.
Troubleshooting: If yield is low, first test GFP alone to confirm system works. If GFP works but membrane protein does not, try:
- Adding lipids during synthesis rather than after.
- Using a different expression tag (e.g., Mistic) that promotes membrane integration.
- Swapping to a eukaryotic system (e.g., insect cell lysate) that naturally processes membrane proteins.
6. Low yield of target protein – three possible reasons and troubleshooting
| Reason | Troubleshooting strategy |
|---|---|
| Incomplete energy regeneration | Increase 3‑PGA or PEP concentration (e.g., from 300 mM to 500 mM). Add creatine phosphate/creatine kinase as a secondary system. |
| RNase contamination | Add more murine RNase inhibitor (e.g., double the amount). Use nuclease‑free water and filter‑sterilised tips. Prepare fresh extract with added RNase inhibitors. |
| Poor template DNA quality or incorrect promoter | Purify plasmid with endotoxin‑free kit. Sequence the T7 promoter and ribosome binding site. Use a linear PCR product with T7 promoter as positive control. Test a known good template (e.g., deGFP) to verify extract activity. |
Expected Results (Theoretical)
A dose‑response curve of IPTG vs. GFP fluorescence would show:
- Basal leakage (0 IPTG + DNA) – low but detectable (if included; our table lacks that control, but typically it exists).
- Sigmoidal increase from 0.2X to 0.4X, plateauing at 0.8X (full induction).
- Positive control (constitutive GFP) gives maximum signal.
- Negative control (no DNA) gives background autofluorescence.
An example plot (not generated physically) would be attached here. Fold change between 0.2X and 0.8X IPTG would be ~3‑5×.
Final Note
All the above is a theoretical exercise. No physical TXTL reactions were assembled, run, or measured. The protocol and answers are based on the provided materials and standard cell‑free literature.