Protocol

1. Medium and reagent preparation

This section provides detailed methods for preparing various media and reagents required for constructing and verifying engineered strains. All procedures must be conducted in a clean environment. For liquid compositions, ultrapure water (18.2 MΩ·cm) should be used, and aseptic techniques must be strictly followed.

1.1. LB Medium

Reagents Required: Tryptone, yeast extract, sodium chloride, agar powder (for solid medium), deionized water, sodium hydroxide pellets, or concentrated hydrochloric acid (for pH adjustment).

LB Liquid Medium:

(1) Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride into an appropriate beaker or Erlenmeyer flask.

(2) Add approximately 800 mL of deionized water and stir with a magnetic stirrer until fully dissolved.

(3) Make up the volume to 1 L with deionized water.

(4) Measure the solution's pH using a pH meter and adjust it to 7.0–7.4 using a small amount of NaOH or HCl.

(5) Aliquot the prepared medium into suitable containers (e.g., test tubes or flasks) and seal them properly.

(6) Sterilize by autoclaving at 121°C for 20 minutes. After sterilization, store at room temperature or in a refrigerator at 4°C for later use.

LB Solid Medium:

(1)Follow steps 1–4 of the liquid LB protocol, but add 15 g of agar powder before making up the volume.

(2) After volumetric adjustment, proceed to autoclave sterilization.

(3) After sterilization, cool the medium to around 55–60°C (warm to the touch but not hot). Add appropriate antibiotics or other heat-sensitive additives as required, then mix gently without introducing bubbles.

(4) Pour the medium into sterile petri dishes in the laminar hood (approximately 20–25 mL per dish), cover the plates, and leave a slight gap for vapor to disperse.

(5) Once solidified, invert the plates, seal the edges with parafilm, and store them in a 4°C refrigerator.

1.2. Antibiotic Stock and Working Solutions

Reagents Required: Ampicillin sodium salt, kanamycin sulfate, chloramphenicol, deionized water, 0.22 μm sterile filter membranes, and syringes.

Ampicillin (100 mg/mL Stock):

(1) Weigh 1 g of ampicillin sodium salt and dissolve it in 8 mL of deionized water.

(2) Stir until completely dissolved, then make up the volume to 10 mL.

(3) Filter sterilize using a 0.22 μm filter and aliquot (e.g., 1 mL per tube) into sterile centrifuge tubes in the laminar hood.

(4) Store at -20°C protected from light. Working concentration: 100 μg/mL.

Kanamycin (50 mg/mL Stock):

(1) Weigh 0.5 g of kanamycin sulfate and dissolve it in 8 mL of deionized water.

Proceed with steps similar to those for ampicillin preparation. Working concentration: 50 μg/mL.

Chloramphenicol (34 mg/mL Stock):

(1) Weigh 0.34 g of chloramphenicol powder and dissolve it in 8 mL of absolute ethanol (due to its poor water solubility).

(2) Stir to dissolve, then make up the volume to 10 mL with ethanol.

(3) No filtration is necessary. Aliquot in a sterile environment.

(4) Store at -20°C protected from light. Working concentration: 25 μg/mL.

1.3. Bile Salt Solution

Reagents Required: Bile salt powder, deionized water, 0.22 μm sterile filter membranes, and syringes.

(1) Weigh 10 g of bile salt powder into a sterile container.

(2) Add approximately 80 mL of deionized water and dissolve using a magnetic stirrer or vortex mixer.

(3) Make up the volume to 100 mL, preparing a 10% (w/v) stock solution.

(4) Filter sterilize using a 0.22 μm filter and aliquot in the laminar hood.

(5) Store at 4°C protected from light. Add to culture media aseptically as needed to reach the required final concentration (e.g., 0.1%, 0.2%, 0.5%).

1.4. Reagents for Electrophoresis and Molecular Cloning

Reagents Required: Tris, acetic acid, disodium ethylenediaminetetraacetic acid (EDTA), agarose, GoldView nucleic acid stain (or alternatives like GelRed), DNA loading buffer, DNA molecular weight markers.

50× TAE Electrophoresis Buffer Stock:

(1) Weigh 242 g of Tris, 37.2 g of Na₂EDTA·2H₂O, and place into a 1 L beaker.

(2) Add ~800 mL of deionized water and stir until dissolved.

(3) Add 57.1 mL of glacial acetic acid and mix thoroughly.

(4) Make up the volume to 1 L with deionized water and store at room temperature. Dilution to working concentration: 1× TAE.

1% Agarose Gel:

(1) Weigh 1 g of agarose powder and place into a 250 mL Erlenmeyer flask.

(2) Add 100 mL of 1× TAE electrophoresis buffer.

(3) Heat in a microwave until the agarose completely dissolves, swirling periodically.

(4) Cool to ~60°C and add a nucleic acid stain (e.g., 5 μL of GoldView). Mix gently and pour into a gel caster with a comb.

(5) Let the gel solidify at room temperature (~30 minutes) before use.

1.5. Competent Cell Preparation and Transformation Reagents

Reagents Required: Rubidium chloride, calcium chloride, glycerol, glucose, magnesium chloride, magnesium sulfate, deionized water, 0.22 μm filters.

SOC Recovery Medium:

(1) Prepare: Dissolve ingredients (tryptone 2%, yeast extract 0.5%, NaCl 10 mM, KCl 2.5 mM) in deionized water and autoclave.

(2) Once cooled, aseptically add 20 mM glucose (filter sterilized) and 10 mM MgCl₂ or MgSO₄ solutions.

RbCl Solutions (For High-Efficiency Competent Cells):

(1) Solution I (100 mM RbCl, 50 mM MnCl₂, 30 mM KAc, 10 mM CaCl₂, 15% Glycerol, pH 5.8): Prepare by accurate weighing. Adjust pH with acetic acid, sterilize, and pre-chill at 4°C.

(2) Solution II (10 mM RbCl, 10 mM MOPS, 75 mM CaCl₂, 15% Glycerol, pH 7.0): Prepare similarly, adjusting pH with NaOH.

0.1 M CaCl₂ Solution (For Simplified Competent Cell Preparation):

(1) Weigh 1.11 g of anhydrous CaCl₂ and dissolve in 80 mL of deionized water.

(2) Make up the volume to 100 mL.

(3) Filter sterilize with a 0.22 μm filter, aliquot, and pre-chill at 4°C.

2. Bacterial culture and chassis cell adaptability verification

This section aims to verify the viability and growth capacity of the selected chassis cell, Lactobacillus casei (L. casei), in a simulated intestinal environment (with bile salts). The results will lay the foundation for subsequent genetic engineering modifications.

2.1. Experimental Reagents

(1) Strains:

• Wild-type Lactobacillus casei (frozen glycerol stock or streaked agar plate).

(2) Culture Media:

• LB liquid medium (antibiotic-free).
• LB solid medium (antibiotic-free).

(3) Reagents:

• Sterile physiological saline (0.85% NaCl) or phosphate-buffered saline (PBS).
• 10% (w/v) sterile-filtered bile salt stock solution.
• Sterile glycerol (for preparing glycerol stock culture).

(4) Consumables and Equipment:

• Sterile test tubes, Erlenmeyer flasks.
• Sterile inoculation loops and glass spreaders.
• Sterile pipette tips.
• Incubator (set to 37°C).
• Shaking incubator (for liquid culture).
• Spectrophotometer.
• Laminar flow hood or biosafety cabinet.

2.2. Experimental Procedures

Stage 1: Strain Resuscitation and Seed Culture Preparation

Streak Plate Resuscitation:

(1) In a sterile environment, use a sterile inoculation loop to pick a small amount of frozen glycerol stock or a single colony from the original plate.

(2) Perform a three-zone streak on a pre-warmed LB solid agar plate to obtain isolated single colonies.

(3) Invert the plate and incubate it at 37°C for 24–48 hours.

Seed Culture Preparation:

(4) Pick a freshly formed, well-shaped, and edge-smooth single colony from the streaked plate.

(5) Inoculate the colony into a sterile test tube or small Erlenmeyer flask containing 5–10 mL of LB liquid medium.

(6) Incubate at 37°C while shaking at 150 rpm (or keep stationary for static culture) for 12–16 hours until the medium becomes visibly turbid (OD₆₀₀ ≈ 1.0). This serves as the seed culture.

Stage 2: Bile Salt Tolerance Test - Liquid Culture Analysis

Preparation of Bile Salt-Supplemented Medium:

(1) Prepare sterile Erlenmeyer flasks, each containing a fixed volume of LB liquid medium.

(2) Using sterile techniques, add the required amount of 10% bile salt stock solution to achieve final concentrations of 0% (control), 0.1%, 0.2%, and 0.5%.

(3) Mix thoroughly by gently swirling.

Inoculation and Cultivation:

(1) Inoculate the pre-prepared seed culture into bile salt-supplemented LB media at a 1:100 dilution ratio (e.g., 100 μL seed culture into 10 mL medium).

(2) Set up at least two parallel replicates for each bile salt concentration.

(3) Place the flasks into a shaking incubator at 37°C, shaking at 200 rpm.

Growth Curve Plotting:

(1) At time intervals of 0, 2, 4, 6, 8, and 24 hours, take samples from each flask.

(2) Measure the optical density at 600 nm (OD₆₀₀) using a spectrophotometer, using the respective uninoculated medium at the same concentration as a blank control.

(3) Record the OD₆₀₀ values and plot growth curves with time on the x-axis and OD₆₀₀ (mean value) on the y-axis for each bile salt concentration.

Stage 3: Bile Salt Tolerance Test - Solid Culture Analysis

Preparation of Bile Salt-Supplemented Agar Plates:

(1) Autoclave LB solid medium and cool to approximately 55°C.

(2) Add the desired volume of 10% bile salt stock solution into the medium under sterile conditions to prepare final concentrations of 0%, 0.1%, 0.2%, and 0.5%.

(3) Mix quickly and pour the medium into sterile Petri dishes to form solid agar plates.

Bacterial Dilution and Plate Spreading:

(1) Dilute the seed culture in sterile physiological saline to prepare a series of 10-fold dilutions, selecting an appropriate dilution to achieve 30–300 CFU per plate.

(2) Pipette 100 μL of the diluted bacterial suspension onto the center of each agar plate with the corresponding bile salt concentration.

(3) Use a sterile spreader to evenly distribute the bacterial suspension across the agar surface.

Incubation and Observation:

(1) Allow the liquid on the plate to be absorbed.

(2) Invert the plates and incubate them at 37°C for 24–48 hours.

(3) Observe and compare the colony size, morphology, and density across different bile salt concentrations.

(4) For countable plates (30–300 CFU/plate), count the colonies and calculate the viable bacterial concentration in the original seed culture (CFU/mL).

3. Plasmid Construction and Amplification

This section provides a detailed description of the process for cloning target genes (e.g., accA, OLE1, DEFB4A) into specific plasmid backbones (e.g., pMG36e, PNZ8048, PIP501) and amplifying them in bacterial hosts.

3.1. Plasmid Construction - Restriction Digestion and Ligation

Double Digestion of Plasmid Backbone and Insert Fragment

Reaction System (for a 50 μL total volume):

• Plasmid DNA or PCR product: 1–2 μg
• 10× FastDigest Green Buffer: 5 μL
• Restriction Enzyme I (e.g., BamHI): 1 μL
• Restriction Enzyme II (e.g., XhoI): 1 μL
• Sterile ultrapure water: To 50 μL total

Protocol:

(1) Assemble the reaction mixture in a sterile PCR tube in the order listed above, mixing gently by pipetting.

(2) Incubate the reaction mixture in a 37°C water bath or metal block for 30–60 minutes.

(3) After digestion, take 5 μL of the product and perform agarose gel electrophoresis to verify successful digestion.

Purification and Recovery of DNA Fragments

(1) Load the remaining digested reaction mixture into wells of an agarose gel and separate the DNA fragments via electrophoresis.

(2) Using a UV gel imaging system, locate the desired DNA bands (correct linearized plasmid backbone and insert fragments) and excise them with a sterile scalpel, minimizing extra gel.

(3) Purify and recover the DNA using an agarose gel extraction kit following the manufacturer's instructions:

• Weigh the gel slices.
• Dissolve the gel in Binding Buffer at 65°C until the gel completely melts.
• Transfer the solution into a spin column and centrifuge.
• Wash the column twice with Wash Buffer and air dry by centrifugation.
• Elute the DNA with Elution Buffer or sterile water and collect the eluate.

(4) Measure the DNA concentration and purity with a micro-volume spectrophotometer.

Ligation Reaction

Reaction System (for a 10–20 μL total volume):

• Linearized plasmid backbone: ~50 ng (Recommended molar ratio: vector:insert = 1:3 to 1:7)
• Insert fragment DNA: X μL (adjusted to achieve the desired molar ratio)
• 10× T4 DNA Ligase Buffer: 1–2 μL
• T4 DNA Ligase: 1 μL (e.g., 5 Weiss U/μL)
• Sterile ultrapure water: Add to 10–20 μL

Protocol:

(1) Mix the reaction components gently and briefly centrifuge to collect drops.

(2) Incubate at 16°C in a water bath or metal block overnight (12–16 hours). For high-efficiency systems, incubation can be shortened to 1–2 hours.

3.2. Plasmid Amplification - Transformation and Culture

Chemical Transformation

(1) Take a tube of chemically competent cells (50–100 μL) from the –80°C freezer and thaw on ice for 5–10 minutes.

(2) Add 5–10 μL of the ligation product (or ~10 ng purified plasmid for a positive control, and use no DNA for a negative control) into the thawed cells. Flick the tube gently to mix without vortexing. Incubate on ice for 30 minutes.

(3) Heat shock the cells at 42°C for exactly 90 seconds in a pre-heated water bath. Immediately place the tube back on ice for 2–5 minutes.

(4) Add 500–900 μL of pre-warmed antibiotic-free LB medium to the tube.

(5) Incubate the tube in a shaking incubator at 37°C and 150–200 rpm for 45–90 minutes to allow the bacteria to recover and express the antibiotic resistance gene.

Plating and Primary Screening of Positive Clones

(1) Briefly centrifuge the recovered bacterial suspension to concentrate the cells, discard part of the supernatant, and resuspend the remaining cells (~100–150 μL).

(2) Spread the resuspended cells evenly onto LB selection plates containing the appropriate antibiotic (e.g., Ampicillin, Kanamycin, or Chloramphenicol) using a sterile spreader.

(3) Allow the liquid to absorb completely, then invert the plates and incubate them at 37°C for 12–16 hours.

(4) After incubation, pick individual well-isolated colonies and inoculate them into 5 mL of LB liquid medium with the corresponding antibiotic.

(5) Grow cultures at 37°C and 220 rpm in a shaking incubator overnight (12–16 hours).

3.3. Plasmid Extraction and Verification

Miniprep to Extract Plasmid DNA

Use a miniprep kit to extract plasmid DNA from 1.5–5 mL of overnight bacterial culture:

• Centrifuge the culture to collect the bacterial pellet.
• Resuspend the pellet in Resuspension Buffer.
• Lyse the cells by adding Lysis Buffer and mix gently until the solution becomes clear and viscous.
• Neutralize with Neutralization Buffer to form precipitates.
• Centrifuge and load the supernatant onto a spin column.
• Wash the column twice with Wash Buffer.
• Elute the plasmid DNA with Elution Buffer or sterile water and store at –20°C.

4. Functional validation analysis

This section aims to systematically validate the designed functions of the engineered bacteria, including target protein expression, synthesis of metabolic products, and the reliability of the biosafety system.

4.1. Protein Level Validation: Western Blot

Experimental Reagents:

• Samples:
  • Induced engineered bacteria (+0.1% bile salts) and non-induced engineered bacteria (no bile salts).
  • Wild-type bacteria as controls.
• Cell Lysis Buffer:
  • RIPA buffer containing PMSF or other protease inhibitors.
• Electrophoresis Reagents:
  • 4× Protein loading buffer.
  • SDS-PAGE precast gel or gel preparation reagents (Tris-HCl, Acrylamide-Bis, SDS, APS, TEMED).
  • Protein molecular weight marker.
  • 1× Electrophoresis running buffer.
• Transfer Reagents:
  • PVDF membrane or NC membrane.
  • 1× Transfer buffer.
  • Methanol.
• Immunodetection Reagents:
  • Blocking solution: 5% skim milk or BSA dissolved in TBST buffer.
  • Primary antibody: Mouse anti-His-tag monoclonal antibody.
  • Secondary antibody: HRP-conjugated goat anti-mouse IgG.
  • Wash buffer: 1× TBST buffer.
  • Substrate: ECL chemiluminescence kit.

Procedure:

Protein Sample Preparation:

(1) Collect 1-5 mL bacterial culture and centrifuge to discard the supernatant.

(2) Wash the bacterial pellet once with PBS and resuspend in an appropriate amount of RIPA lysis buffer. Incubate on ice for 30 minutes with intermittent vortexing.

(3) Centrifuge at 4°C, 12,000 rpm for 15 minutes and collect the supernatant as the total protein sample.

(4) Mix the protein supernatant with 4× protein loading buffer at a 3:1 ratio and heat in a boiling water bath for 10 minutes to denature proteins.

SDS-PAGE:

(1) Load the prepared protein samples and molecular weight marker into the wells of an SDS-PAGE gel.

(2) Run electrophoresis at an initial voltage of 80V, switching to 120V after the samples enter the separation gel, and continue until the bromophenol blue dye reaches the gel bottom.

Membrane Transfer:

(3) Perform wet transfer. Assemble a "sandwich" structure with the gel, pre-activated PVDF membrane (activated with methanol), filter paper, and sponge soaked in transfer buffer.

(4) Transfer proteins at 4°C using a constant current of 250 mA for 60-90 minutes.

Immunoblotting and Detection:

(1) After transfer, place the PVDF membrane in 5% skim milk blocking solution and incubate at room temperature for 1 hour.

(2) Wash the membrane three times with TBST buffer, 5 minutes each.

(3) Incubate with the primary antibody solution (diluted according to the manufacturer's instructions) overnight at 4°C or at room temperature for 2 hours.

(4) Wash the membrane three times with TBST buffer for 10 minutes each.

(5) Incubate with the HRP-conjugated secondary antibody (diluted according to the manufacturer's instructions) at room temperature for 1 hour.

(6) Wash thoroughly with TBST, then add an equal mixture of ECL substrate A and B solution to the membrane.

(7) Expose the membrane in a chemiluminescence imaging system and capture the image.

4.2. Metabolite Level Validation: Liquid Chromatography Analysis

Experimental Reagents:

• Samples:
  • Culture supernatants from different engineered bacteria and control strains (centrifuged and filtered through 0.22 μm membranes).
• Standards:
  • Short-chain fatty acids (acetic acid, propionic acid, butyric acid) and oleic acid standards.
• Mobile Phase:
  • Acidic aqueous solution (e.g., 0.005 M H₂SO₄) prepared based on chromatography requirements.
• Consumables:
  • HPLC injection vials and 0.22 μm syringe filters compatible with aqueous samples.

Procedure:

Sample Preparation:

(1) Centrifuge collected culture supernatants at 4°C, 12,000 rpm for 10 minutes, and filter the supernatants through 0.22 μm syringe filters. Transfer into HPLC vials.

Standard Curve Preparation:

(1) Prepare a series of standard solutions with known concentrations of each metabolite and analyze using the same chromatographic conditions to generate standard curves.

Chromatographic Conditions (Example, Subject to Optimization):

• Column:
  • C18 reverse-phase column (for oleic acid) or a specialized organic acid analysis column (e.g., Aminex HPX-87H for short-chain fatty acids).
• Detector:
  • UV detector (for oleic acid, detection at ~205 nm) or refractive index detector (RID, for general applications).
• Mobile Phase and Flow Rate:
  • Adjust according to the selected column and method.
• Column Temperature:
  • 30-50°C.

Sample Analysis:

(1) Inject prepared samples and standards sequentially and record chromatograms.

(2) Identify metabolites by comparing the retention times of sample peaks to those of standards. Quantify concentrations using peak areas and standard curves.

4.3. Biosafety System Validation: Bactericidal Curve Assay

Experimental Reagents:

• Strains:
  • Engineered bacteria carrying the Hok/Sok system.
• Culture Media:
  • LB broth with 0.1% bile salts ("intestinal environment").
  • LB broth without bile salts ("non-intestinal environment").
• Reagent:
  • Sterile PBS.

Procedure:

Pre-culture and Induction:

(1) Inoculate engineered bacteria into LB broth with 0.1% bile salts and incubate at 37°C until mid-log phase (OD₆₀₀ ≈ 0.6). This simulates the intestinal environment where the biosafety system is suppressed.

Environmental Shift:

(2) Harvest a suitable volume of bacterial culture by centrifugation and collect the pellet.

(3) Wash the cells gently twice with sterile PBS to remove residual bile salts.

(4) Resuspend the pellet in an equal volume of fresh LB broth without bile salts. Mark this time point as t=0.

Sampling and Colony Counting:

(1) At various time points, take samples from the culture.

(2) Perform serial 10-fold dilutions in sterile PBS.

(3) Plate 100 μL from suitable dilutions onto LB agar plates without bile salts but with antibiotics to select for the engineered strain.

(4) Incubate plates at 37°C for 24-48 hours.

Data Recording and Analysis:

(1) Count colonies on plates with 30-300 colonies and calculate CFU/mL for each time point using dilution factors.

(2) Plot a killing curve with time as the x-axis and log(CFU/mL) as the y-axis.