Experiments

Laboratory protocols, procedures and experimental design

Introduction

Our Experimental Approach

Our experimental approach follows rigorous scientific methodology, ensuring reliable and reproducible results in our synthetic biology research. Below you will find detailed protocols for key laboratory procedures used throughout our project.

Electrocompetent Cells

Preparation Protocol

Aim

To prepare highly competent E. coli cells for efficient DNA uptake via electroporation while maintaining cell viability and sterility.

Materials and Reagents

LB medium
Sterile MilliQ water
10% glycerol
E. coli strain
50mL tubes
Ice

Procedure

Day 0
  1. Inoculate 5mL LB medium with a single colony of E. coli.
  2. Incubate overnight at 37°C, 250 rpm.
Day 1
  1. Inoculate 1000mL pre-warmed LB with 5mL of overnight culture.
  2. Incubate at 37°C, 250 rpm for ~5 hours until OD₆₀₀ = 0.5–0.7.
  3. Distribute culture into six 50mL centrifuge tubes.
  4. Cool tubes on ice for 15–60 minutes.
  5. Centrifuge at 2000 × g (4500 rpm) for 10 min at 4°C.
  6. Discard supernatant, resuspend in 50mL ice-cold MilliQ water.
  7. Centrifuge again and discard supernatant.
  8. Wash pellets in 50mL of 10% glycerol for 30 min to 1 hour.
  9. Centrifuge and discard supernatant.
  10. Resuspend in 3mL total 10% glycerol (~500µL per tube).
  11. Aliquot 100µL per tube into sterile microcentrifuge tubes.
  12. Store at –80°C (or flash freeze with liquid nitrogen).

Electroporation

Transformation Protocol

Aim

To transform E. coli cells by electroporation, efficiently introducing plasmid DNA under sterile conditions for applications in cloning or synthetic biology.

Materials and Reagents

Electrocompetent cells
Plasmid DNA (<100 ng)
LB medium (sterile)
Electroporation cuvettes
Electroporator (Ec2)
LB agar plates

Procedure

  1. Thaw electrocompetent cells on ice.
  2. Add 2 µL plasmid DNA (<100 ng) to 50 µL thawed cells.
  3. Mix gently by pipetting up and down.
  4. Pipette into pre-chilled electroporation cuvette.
  5. Ensure suspension is at bottom, wipe condensation.
  6. Insert cuvette, press "Pulse" (Ec2: 12.5 kV/cm).
  7. Immediately add 950 µL LB, mix by pipetting.
  8. Transfer to sterile 1.5 mL tube.
  9. Incubate 2 hours at 37°C with shaking.
  10. Plate 50 µL on LB agar with antibiotic.

Enzymatic Digestion

Restriction Enzyme Protocol

Aim

Cut vector and insert with NcoI and SacI to generate compatible cohesive ends for directional cloning.

Materials

Vector DNA (500–1000 ng)
PCR product (5–10 µL)
NcoI and SacI enzymes
Tango 10X Buffer
Nuclease-free water
Microcentrifuge tubes

1. Vector Digestion

Component Volume (20 µL)
Nuclease-free water To final volume
Tango 10X buffer 2 µL
DNA (purified plasmid) 500 ng
Enzyme (10 U/µL) 1 µL
Enzyme (10 U/µL) 1 µL

Incubate at 37°C for 3 hours, then purify.

2. Insert Digestion

Component Volume (20 µL)
Nuclease-free water 6 µL
Tango 10X buffer 2 µL
PCR product 10 µL
Enzyme (10 U/µL) 1 µL
Enzyme (10 U/µL) 1 µL
💡 Tips for Success
  • Enzyme ≤10% of reaction volume
  • Use compatible buffer (Tango 10X)
  • Mix gently, avoid vortexing
  • Add 3–6 extra nucleotides at 5' primer ends

Ligation Protocol

DNA Assembly

Aim

Join DNA insert and vector using T4 DNA Ligase to generate a recombinant plasmid for transformation.

Materials

Vector DNA (50–100 ng)
Insert DNA (3:1 ratio)
T4 DNA Ligase
10X Ligase Buffer (ATP)
Nuclease-free water
Microcentrifuge tubes

Protocol Setup

Component Volume (20 µL)
Nuclease-free water To final volume
10X T4 Ligase Buffer 2 µL
Vector DNA (50–100 ng) [ ] µL
Insert DNA (3:1 molar ratio) [ ] µL
T4 DNA Ligase (5 U/µL) 1 µL

Incubation: Cycle 30s at 10°C / 30s at 30°C for 12–16 hours (overnight)

💡 Tips for Success
  • Maintain 3:1 to 5:1 insert:vector molar ratio
  • Use fresh ATP-containing buffer
  • Purify fragments before ligation
  • Include vector-only negative control

Fluorescence and Absorbance Calibration

InterLab 2023 Calibration Protocol

To ensure our experimental fluorescence data were accurate and comparable to measurements from other laboratories and instruments, we followed the 2023 iGEM InterLab Multicolor Fluorescence per Particle Calibration Protocol.

This protocol standardizes fluorescence readings by converting arbitrary plate reader units into absolute fluorescence values using fluorescein, sulforhodamine 101, cascade blue, and NanoCym silica nanoparticles as calibrants.

Calibration was performed under the same conditions as our characterization experiments (same plate type, volume, and reader settings), guaranteeing data consistency across the DBTL cycle.

Full Protocol

You can access the full step-by-step protocol here:

InterLab 2023 Calibration Protocol

In our DBTL characterization of the pLux promoter (BBa_K2656028), accurate fluorescence measurements were essential for parameter identification and model validation.

By performing the InterLab calibration, we ensured that GFP fluorescence values obtained from our plate reader could be expressed in Molecules of Equivalent Fluorescein (MEFL), allowing a direct comparison between simulations and experiments.

This step also made our data compatible with previous iGEM characterizations and standardized the output for model-driven learning within our Engineering cycle, where calibrated experimental data guided the parameter optimization of the LuxR–AHL promoter system.

Equipment and Measurement Settings

All fluorescence and absorbance readings were taken using a 96-well black plate with a transparent flat bottom, filled with 200 µL per well.

Measurements were carried out using a microplate reader configured as follows:

Excitation: 488 nm
Emission: 530 nm
Bandpass: 30 nm
Temperature: 37 °C
Shaking mode: Double orbital, 230 rpm
Sampling frequency: Every 5 minutes for 20 hours

These same parameters were used for both calibration and experimental measurements, ensuring reproducibility and reliable conversion of fluorescence data into standard units.

Cell Measurement Protocol – InterLab 2023

Standardized Fluorescence Measurements

For the fluorescence and absorbance measurements of our own devices, we followed the 2023 iGEM InterLab Study – Experiment 3: Cell Measurement Protocol (Growth in Test Tubes vs. 96-Well Plates), applying only the 96-well plate format.

Although this protocol was originally designed to compare E. coli K-12 DH5-alpha cultures carrying standard iGEM test devices, we used its methodology and measurement conditions to ensure standardized, reproducible, and comparable fluorescence data for our constructs.

Measurement Conditions

Cultures were grown and monitored in 96-well black plates with flat bottoms, containing 200 µL per well, incubated at 37 °C and 220 rpm under double orbital shaking.

Fluorescence (excitation 488 nm, emission 530 nm) and optical density (OD₆₀₀) were recorded at 0 and 6 hours following the InterLab timing and settings.

By adhering to this validated community protocol, we ensured that all fluorescence data for our pLux promoter DBTL characterization were generated under conditions consistent with global iGEM measurement standards.

Full Protocol

You can access the full step-by-step protocol here:

InterLab 2023 Experiment 3 Protocol
Why This Experiment Matters

Using the InterLab Experiment 3 plate reader protocol as our measurement standard ensured that our fluorescence and absorbance data were quantitatively reliable and inter-lab comparable.

This standardization was essential for the Learn stage of our DBTL characterization of the pLux promoter (BBa_K2656028), allowing our parameter estimation and model validation to be performed using globally reproducible methods.