Synthetic Biology & Quantitative Assays
Cell-Free TXTL Riboswitch Assay Optimization
Our synthetic circuit validation employs the myTXTL Pro 2.0 In Vitro Expression System to study riboswitch-mediated gene regulation. These protocols detail the template linearization, concentration normalization, and the rigorous optimization of the reaction stoichiometry for the toehold switch sensor against the target IL7R RNA trigger. Maintaining optimal DNA:RNA molar ratios and reaction capacity is critical for achieving high fold-induction and accurate kinetic characterization.
Assay Procedure Modules
Concentration Normalization
Quantification and concentration adjustment of linear DNA template (Toehold-Reporter) and RNA trigger oligo (sIL7R) to ensure precise input molarity. This is Protocol 1.0.
- Linear DNA Stock: 160 nM.
- RNA Working Stock: 20 uM.
- Spectrophotometric verification (A260/A280 and A260/A230) is mandatory.
TXTL Stoichiometry Assembly
Implementation of the 12 uL reaction with modified DNA volume to preserve the TXTL energy capacity despite the addition of the 1.0 uL RNA oligo. This is Protocol 2.0.
- DNA concentration in reaction: ~5 nM.
- RNA concentration in reaction: ~1.67 uM (assuming 20 uM stock used).
- Inclusion of essential biochemical controls for specificity and leakage.
Reporter Kinetic Assays (Trials 1-10)
This section contains three distinct kinetic protocols: AmilCP Pilot (3.0), GFP Optimization (4.0), and Standard GFP Replicates (5.0), detailing the specific readout settings for each trial phase.
- Protocol 3.0: Absorbance (A588) for Trial 1.
- Protocol 4.0: Fluorescence (488/509 nm) for Trial 2 (Optimization).
- Protocol 5.0: Fluorescence (488/509 nm) for Trials 3-10 (Validation).
Detailed Protocols
Protocol 1.0: Linear DNA and RNA Template Concentration Normalization
Purpose: To prepare high-concentration stock solutions for all DNA and RNA templates. This normalization ensures the final reaction concentration of DNA remains at approximately 5 nM after volumetric adjustments, thereby minimizing TXTL inhibition due to template competition or DNA concentration effects.
Part A: Linear DNA Template Stock (Target 160 nM)
Quantitative Rationale
- Target Stock: 160 nM is required so that a 1.5 uL addition to the 12 uL reaction yields the desired ~5 nM final DNA concentration (160 nM x (1.5 / 12) ~ 20 nM theoretical final concentration, which accounts for the 2.5 uL standard use at 96 nM target).
- Expected Mass Conc.: For a 970 bp linear template, 160 nM equates to ~101 ng/uL (based on MW dsDNA ~ 650 g/mol/bp).
- Quality Control: A260/A280 ratio must be 1.8-2.2 (protein/phenol purity); A260/A230 ratio must be 2.0-2.3 (salt/organic purity).
Reconstitution Procedure
- Centrifuge dry DNA tube (>3000 x g) to consolidate material.
- Reconstitute by adding 8.64 uL nuclease-free water to achieve 160 nM stock.
- Vortex briefly, then thermal incubation at 50 deg C for 15-20 min to ensure complete dissolution.
- Confirm concentration and purity via Nanodrop spectrophotometer. Store on ice.
Part B: RNA Oligo Stock Preparation (Target 20 uM Working)
Concentration Details
- Initial Resuspension: Dry RNA oligo is reconstituted to a 200 uM master stock. (e.g., 67 nmol of RNA -> 335 uL water).
- Working Stock: A 10-fold dilution is made to prepare the 20 uM working stock (2 uL of 200 uM + 18 uL water).
- Final Reaction Conc.: Addition of 1.0 uL of 20 uM stock to 12 uL total reaction volume results in an ~1.67 uM RNA concentration, ensuring a massive molar excess relative to the DNA template for efficient toehold activation.
Reconstitution Procedure
- Reconstitute dry RNA oligo to the 200 uM master stock using the nmol x 10 = uL water rule.
- Enhance dissolution: Vortex, followed by brief heat incubation at 50 deg C - 55 deg C for 3-5 min, vortex again, and quick-spin.
- Prepare 20 uM working stock; aliquot using RNase-free materials. Store master stock at -20 deg C.
Protocol 2.0: Cell-Free TXTL Reaction Assembly and Control Strategy (12 uL Volume)
Purpose: To assemble the TXTL reactions under isothermal conditions (27 deg C). The DNA volume is reduced from the standard 2.5 uL to 1.5 uL to maintain the total 12 uL reaction volume after the required 1.0 uL RNA oligo addition, preserving the stoichiometric integrity of the Master Mix components.
Optimized 12 uL Mix Composition
- Pro Master Mix: 9.0 uL (Core machinery, ATP regeneration)
- Pro Helper Plasmid: 0.5 uL (Enhances expression of essential factors)
- Template DNA (160 nM stock): 1.5 uL
- RNA Oligo (20 uM working): 1.0 uL
- Total Volume: 12.0 uL
Assembly Procedure
- Pre-equilibrate plate reader or incubator to the reaction temperature (27 deg C).
- Thaw and immediately transfer all components to ice. Master Mix must be briefly spun down and mixed well by pipetting just prior to use.
- Assemble reactions by sequentially adding Master Mix, Helper Plasmid, Template DNA, and finally RNA Oligo or Water.
- Apply a quick vortex and mini-centrifuge (~1 s) to ensure mixture homogeneity and liquid collection at the well bottom.
- Immediately load sealed plate into the 27 deg C reader for kinetic monitoring.
Rigorous Control Strategy (12 uL per well)
| Control Condition / Purpose | Template DNA | RNA Oligo / Water | Biochemical Validation |
|---|---|---|---|
| Experimental (Targeted Activation) | 1.5 uL Toehold DNA | 1.0 uL sIL7R RNA Trigger | Measure Fold-Induction and Activation Rate |
| Neg Control (Non-Cognate RNA) | 1.5 uL Toehold DNA | 1.0 uL Randomized RNA | Confirms Sequence-Specificity of the toehold switch |
| Neg Control (No RNA / Leakage) | 1.5 uL Toehold DNA | 1.0 uL Nuclease-Free Water | Establishes the basal leakage rate of the toehold construct |
| Pos Control (TXTL Functionality) | 2.5 uL deGFP Control DNA | 0.0 uL RNA / Fill with Water | Verifies ribosomal/TXTL energy and enzyme integrity |
Kinetic Assay Suite (Protocols 3.0, 4.0, 5.0)
Protocol 3.0: AmilCP Pilot Assay Kinetic Monitoring (Trial #1)
Purpose: Initial non-fluorescent proof-of-concept for toehold switch activation. This protocol uses the AmilCP chromophore reporter, which is monitored via absorbance to confirm protein expression and functionality before moving to high-throughput GFP screens.
Kinetic Protocol Settings
- Isothermal Control: Reader must maintain 27 deg C throughout the experiment (16-18 hours).
- Kinetics: Continuous reading, e.g., every 10 min.
- Reporter Type: AmilCP (Purple Chromophore).
Spectrophotometric Parameters
- Readout: Absorbance at wavelength 588 nm (peak for the purple chromophore).
- Data Correction: Background signal is subtracted using the Neg Control (No RNA) well.
- Alternative: Snapshot protocol (briefly remove, read, return to 27 deg C) if the reader is non-kinetic.
Protocol 4.0: GFP Dose-Response & Optimization (Trial #2)
Purpose: To determine the optimal DNA:RNA molar ratio that maximizes the signal-to-noise ratio (fold-induction) of the toehold switch. This trial involves setting up a titration of the RNA trigger oligo (e.g., 5, 10, 20, 40 uM stock concentrations) while holding the DNA template concentration constant.
Kinetic Protocol Settings
- Isothermal Control: Reader must maintain 27 deg C throughout the experiment (16-18 hours).
- Kinetics: Continuous reading, e.g., every 3-5 min.
- Reporter Type: deGFP (Enhanced Green Fluorescent Protein).
Spectrophotometric Parameters
- Readout: Fluorescence (Excitation 488 nm, Emission 509 nm).
- Data Correction: Background signal is subtracted using the Neg Control (No RNA) well.
- Initial Rate (v0) calculation is a primary endpoint for optimization.
Protocol 5.0: Standard GFP Kinetic Monitoring (Trials #3-10)
Purpose: To obtain highly repeatable, quantitative kinetic data for the toehold switch using the optimal RNA concentration determined in Protocol 4.0. Trials 3-10 represent the final, validated data set for fold-induction and kinetic analysis.
Kinetic Protocol Settings
- Isothermal Control: Reader must maintain 27 deg C throughout the experiment (16-18 hours).
- Kinetics: Continuous reading for 16-18 hours, with a high frequency read (e.g., every 3-5 min).
- Sample Handling: Plate must be optically clear-sealed and quick-spun immediately before loading.
Spectrophotometric Parameters
- Readout: Fluorescence (Excitation 488 nm, Emission 509 nm).
- Data Correction: Background signal is subtracted using the Neg Control (No RNA) well.
- Primary Output: Fold-Induction (Signal/Leakage Ratio).
Snapshot Protocol (Non-Kinetic Reader) - Applicable to all Kinetic Protocols (3.0, 4.0, 5.0)
For plate readers lacking isothermal control, a Snapshot Protocol is used, which introduces inherent temporal quantization errors but maintains temperature fidelity:
- Incubate the sealed plate at 27 deg C in a dedicated incubator or thermomixer.
- At predefined timepoints (e.g., every 10 min during the early exponential phase), briefly remove the plate, take the reading, and immediately return the plate to the 27 deg C environment. This minimizes temperature fluctuation stress on the TXTL reaction.
Supporting Documents
Browse the verified protocol PDFs directly below: