Experiments

Overview

The Fleur wet‑lab program developed robust transformation protocols for probiotic Lactobacillus species to enable GLP‑1 expression. We combined systematic protocol optimization with rigorous documentation and iterative troubleshooting to establish reliable workflows that underpin our therapeutic goals.

Experimental Strategy

Research Objectives
  1. Optimize Lactobacillus acidophilus and L. plantarum as GLP‑1 hosts.1
  2. Establish reliable transformation methods for probiotics.1
  3. Systematically optimize parameters for maximal efficiency.1
  4. Implement validation systems for reproducibility.2
Target Organisms
  • Primary: L. acidophilus (GRAS), L. plantarum (GLP‑1 precedent).3
  • Control: E. coli for protocol validation.1

Materials and Methods

Media Preparation
LB Broth

Per liter: 10 g casein peptone, 5 g yeast extract, 10 g NaCl. Dissolve 25 g/L, autoclave 121°C 15 min. For agar, add 15 g agar before autoclave.4

MRS Broth

Per liter: 10 g proteose peptone no.3; 10 g beef extract; 5 g yeast extract; 20 g dextrose; 1 g polysorbate 80; 2 g ammonium citrate; 5 g sodium acetate; 0.1 g MgSO4; 0.05 g MnSO4; 2 g K2HPO4. Suspend 55 g/L, boil 1 min, autoclave 121°C 15 min. For agar, add 15 g agar.4

Antibiotic Stocks

Chloramphenicol: stock 10 mg/mL in ethanol; working 0.5–34 μg/mL; filter sterilize; store −20°C.2

Competent Cell Preparation
Chemically Competent

Key reagents: 1 M KCl, Glucose, MgCl2, MOPS, 0.1 M acetic acid, 1 M KOH. Target OD600 0.659–0.750; ice ≥30 min; 0.5 M sucrose + 10% glycerol modification.1,2

Electrocompetent
  1. Grow to mid‑log (OD600 0.4–0.6); chill.
  2. Spin 4°C, 4000 rpm, 10 min; wash 3× with ice‑cold 10% glycerol.
  3. Resuspend 100–200 μL per aliquot; flash freeze; store −80°C.1
Glycerol Stocks

Prepare 50% glycerol (1:1 with culture) and autoclave; use for long‑term storage.4

Transformation Protocols

Heat Shock (baseline)
  1. Thaw competent cells on ice 20–30 min; warm plates.
  2. Add 1–5 μL DNA to 20–50 μL cells; mix gently; ice 20–30 min.
  3. Heat shock 42°C for 30–60 s; ice 2 min.
  4. Recover in LB/SOC 45 min at 37°C; plate 50 μL and remainder; incubate overnight.2

Optimization outcome: 20 μL cells + 1 μL DNA + 10 μg/mL Cm; however, Lactobacillus transformations were unsuccessful → pivot to electroporation.1

Electroporation
OrganismVoltageCapacitanceResistanceCuvette
E. coli1800 V25 μF200 Ω1 mm
L. plantarum2000 V25 μF400 Ω2 mm
L. acidophilus2000 V25 μF400 Ω2 mm
Optimized Procedure
  1. Pre‑chill new cuvettes; ensure DNA in low‑salt buffer; thaw cells on ice.
  2. Use 100–200 μL cells + 1–3 μL DNA; transfer to chilled cuvette.
  3. Pulsing per species; record time constant and voltage.
  4. Immediate recovery: +500 μL media; shake 1–2 h; plate 25–300 μL at multiple antibiotic levels; incubate 37°C overnight.1
Troubleshooting/Optimization
  • Arcing eliminated by new cuvettes and correct gap settings.
  • Optimal recovery window 1–2 h; >2 h caused lawns; <1 h reduced colonies.
  • Best results: L. plantarum 200 μL cells, 1989 V, 6.9 ms; L. acidophilus 100 μL cells, 1977 V, 8.7 ms.1

E. coli Validation Experiments

Validated electroporation hardware and workflow using fluorescent plasmids (M3, C3). Settings: 2500 V, 2 mm, 250 Ω, 25 μF; OD600 0.695–0.750. Initial lawns indicated over‑recovery; tuning to 1–2 h and fresh competent cells improved outcomes.1

Analytical Methods

Miniprep
  1. Pick single colonies to selective media; grow overnight.
  2. Harvest, alkaline lysis, and quantify DNA.
Restriction Digest

Master mix per sample: 12 μL H2O, 2 μL CutSmart, 0.5 μL ApaI, 0.5 μL HindIII; +5 μL DNA; 37°C for 1–3 h; analyze by gel. Use fresh agarose/TAE to avoid dissolution.1

Gel Electrophoresis

1% agarose in 1× TAE; pour ~60°C; include stain. Replace aged reagents to prevent gel failure.

Colony Screening
  • UV visualization for fluorescence; pick positives.
  • Antibiotic‑based selection at 10 and 34 μg/mL.
  • Extended incubation for slow‑growing Lactobacillus; L. acidophilus prefers 42–50°C.

Quality Control and Validation

Controls
  • Negative: no‑DNA, untransformed on antibiotic, heat‑inactivated DNA.
  • Positive: known plasmid, commercial controls, cjBlue (P2‑G1) kit.1
Documentation
  • Record efficiency, time constants, voltages, counts, morphology, temperature/time optimizations; maintain troubleshooting logs.

Results Summary

Key Achievements
  1. Established electroporation protocols for both target species.
  2. Identified optimal parameters for transformation efficiency.
  3. Implemented comprehensive control/validation procedures.
  4. Built robust troubleshooting methodology.
Critical Success Factors
  • Species‑specific voltages/capacitance; fresh cuvettes; recovery 1–2 h; extended incubation.
Current Status
  • Transformations confirmed by growth/resistance; scale‑up prepared for GLP‑1 expression.3

Future Experimental Directions

  1. GLP‑1 expression validation: Western, ELISA, time‑course.3
  2. Secretion optimization: signal peptides, protease resistance, fermentation.
  3. Functional characterization: GLP‑1R binding, cAMP, stability studies.
  4. Advanced: glucose‑responsive regulation, co‑culture/scale‑up, formulation.3
⁂

References

  1. iGEM‑Summer‑Write‑Up.pdf
  2. Bacterial‑Transformation‑Heat‑Shock.pdf
  3. Fleur‑Summer‑Wet‑Lab‑Protocols‑Outline‑1.pdf
  4. Solutions‑Document.pdf

Additional: AddGene Bacterial Transformation Protocol (2024).