I. Cultivation of Strains
1.1 Basic Cholesterol Medium (BCM)
Materials and Concentrations
BCM Formula:
Casitone: 10g/L
Yeast Extract: 10g/L
Cholesterol: 2g/L (adjust as needed)
Sodium 2-Mercaptoethanesulfonate: 0.5g/L
CaCl2⋅2H2O: 1g/L
Procedure
1. Weigh the ingredients for the BCM formula (excluding cholesterol) according to their proportions and mix them in a conical flask. Top up with ultrapure water to obtain the BCM stock solution.
2. Seal the prepared BCM stock solution in conical flasks and sterilize under high-pressure steam at 121°C for 20 minutes.
3. In aseptic conditions, filter-sterilize the cholesterol stock solution as needed using an ultrafiltration membrane.
4. Add the cholesterol stock solution to the BCM stock solution to achieve the desired concentration, creating the BCM culture medium.
2. M9 Culture Medium
Materials and Concentrations
1 mol/L MgSO4
1 mol/L CaCl2
5×M9 Salt Solution: Na2HPO4•12H2O 4.06 g; KH2PO4 1.96 g; NaCl 0.5 g; NH4Cl 1.0 g; Dissolve in 200 mL of ultrapure water and adjust to the final volume.
0.5 g/L Glucose Solution
Note: Depending on the experiment, add bile salts, cholesterol, or adjust the glucose concentration.
Procedure
1. Prepare a 500 mL system of M9 culture medium: A solution 1 mL; B solution 0.05 mL; C solution 100 mL; D solution 10 mL. Top up with sterile ultrapure water to 500 mL.
2. A solution is 1 mol/L MgSO4; B solution is 1 mol/L CaCl2; C solution is 5×M9 Salt Solution; D solution is 0.5 g/L Glucose Solution.
3. Sterilize solutions A, B, and C at 121°C under high-pressure steam for 20 minutes, and D solution under ultraviolet light for 30 minutes.
4. After sterilization, allow the reagents to cool to near room temperature and mix them according to the M9 culture medium system.
3. MRS Culture Medium
Materials and Concentrations
MRS Meat Broth Powder
Procedure
1. Prepare the MRS culture medium according to the instructions on the MRS meat broth powder, based on the desired volume.
2. Seal the prepared MRS solution in conical flasks and sterilize under high-pressure steam at 121°C for 20 minutes.
4. MRS Solid Identification Culture Medium
Materials and Concentrations
MRS Meat Broth Powder
Sodium taurocholate: 3 g/L
Calcium chloride: 0.37 g/L
Agar: 15g/L
Experimental Procedures
1. Preparation of MRS Solid Identification Culture Medium
Materials and Concentrations
MRS Meat Broth Powder
Sodium taurocholate: 3 g/L
Calcium chloride: 0.37 g/L
Agar: 15g/L
Procedure
1. Prepare the MRS solid identification culture medium according to the instructions on the MRS meat broth powder, based on the specified volume and component ratios.
2. Seal the prepared MRS medium in conical flasks and sterilize it under high-pressure steam at 121°C for 20 minutes. Pour the medium onto Petri dishes as soon as it is cool enough to touch but not solidified.
5. LB Culture Medium
Materials and Concentrations
Pancreatic digest of casein: 10g/L
Yeast extract: 5g/L
Sodium chloride: 10g/L
pH Adjustment
Sodium hydroxide
For Solid Culture Media
Agar: 15g/L
Procedure
1. Prepare the LB culture medium according to the LB formula and top up with ultrapure water.
2. Seal the prepared LB medium in conical flasks and sterilize it under high-pressure steam at 121°C for 20 minutes. If it is a solid culture medium, pour it onto Petri dishes as soon as it is cool enough to touch but not solidified. If it's an ampicillin-containing culture medium, add ampicillin (0.5 μg/mL) just before pouring the plates (be cautious as ampicillin can degrade at high temperatures).
6. Gradient Egg Yolk Culture Medium
Materials
Fresh chicken eggs
LB Culture Medium
Procedure
1. Take three fresh chicken eggs, wash them thoroughly, collect the egg yolks, and remove as much egg white as possible. Then, stir the egg yolks on ice for about 20 minutes until it becomes a uniform paste.
2. Prepare five 100 mL LB culture medium bottles. Add 2 mL, 2 mL, 4 mL, 6 mL, and 8 mL of the egg yolk paste to the bottles individually, shake well (before adding the egg yolks, inoculate the LB culture medium with 100 μL of the preserved bacterial culture, and culture it overnight at 37°C and 150 rpm).
7. Cultivation Conditions
General Cultivation Methods
1. Cultivate in 25 cm² culture flasks (Corning, USA) at 37°C under 5% CO2 conditions.
2. Cultivate in conical flasks at 37°C under 5% CO2 conditions.
3. Cultivate in test tubes at 37°C under 5% CO2 conditions.
4. Cultivate on agar plates at 37°C under 5% CO2 conditions.
5. Cultivation methods include using an incubator or a shaking bed.
Anaerobic Cultivation
1. Statically cultivate bacteria in anaerobic bags with oxygen indicators (Hopebiol, Qingdao Haibo, China).
8. Subculturing, Activation, Expansion, and Preservation of Strains
Subculturing of Strains
Transfer 100 μL of the bacterial culture from 5 mL of culture medium to a new 5 mL culture medium.
Activation of Strains
First, culture the strains overnight.
Transfer 1% of the volume of the new culture medium into the fresh culture medium and culture for 3 hours.
Alternatively, activate strains by transferring 100 μL from frozen stocks into 5 mL of culture medium.
Expansion of Strains
Select a bacterial colony or draw 100 μL of bacterial culture.
Culture in the required culture medium (3-5 mL) at 37°C.
Preservation of Strains
Take the activated bacterial culture.
Prepare a preservation mixture with glycerol (glycerol: bacterial culture = 1:1). Store at -80°C.
II. Construction of Engineered Bacteria
1. Preparation of Competent Cells
Materials
Receptor strain (EcN)
LB culture medium
0.1 M Calcium Chloride
Preservation
30% Glycerol
Procedure
1. Activate the strain by overnight cultivation.
2. Transfer 100 μL of the overnight-activated culture into 100 mL of liquid LB medium and culture for 2 hours until the OD reaches 0.5-0.6. Then, aliquot the culture into 50 mL centrifuge tubes (25 mL each) and pre-cool them at 4°C.
3. Centrifuge at 4000g for 10 minutes at 4°C, discard the supernatant, and add 20 mL of 0.1 M CaCl2 to each tube (5 mL per tube). Incubate in an ice bath for 30 minutes.
4. Centrifuge again, discard the supernatant, and add 1 mL of 15% 0.1 M CaCl2 (4 mL for every 100 mL of initial culture). Distribute into 1.5 mL centrifuge tubes, with each tube containing 100 μL. For preservation of competent cells, add a mixture of calcium chloride and 30% glycerol (1:1) to each tube, then store at -80°C.
2. Plasmid Transformation
Materials
Transformation plasmid
Required culture medium (LB culture medium)
Corresponding antibiotic plates (ampicillin-resistant)
Procedure
1. Add 10 μL of the plasmid to 100 μL of competent cells and incubate on ice for 30 minutes (if transformation efficiency is high, try adding 10 μL of plasmid to 20 μL of competent cells, adjusting the culture medium (LB) accordingly).
2. Incubate at 42°C for 60 seconds, then on ice for 1 minute.
3. Add 1 mL of culture medium and incubate at 37°C for 1 hour.
4. Plate on antibiotic plates (spread 100 μL of bacterial culture).
5. Pick a single colony for expansion in 5 mL of LB culture medium. After expansion, store in glycerol vials for preservation.
III. Detection Methods
1. OPA Method for Cholesterol Detection
Materials and Concentrations
Sample solution
0.1 mg/ml cholesterol working solution
Mixed acid (concentrated sulfuric acid: glacial acetic acid = 1:1)
Methanol
Procedure
- Start by establishing a standard curve. Sequentially take 0 mL-0.4 mL of the cholesterol working solution to create nine gradients. To each gradient, add 4 mL of mixed acid and 0.2 mL of ortho-phthalaldehyde. Use methanol to bring each gradient to a total volume of 4.6 mL. The order of addition is the working solution, methanol, ortho-phthalaldehyde, and mixed acid. After adding all components, mix well, allow to stand for 20 minutes, and then measure the absorbance at 550 nm.
- For sample testing, dilute the sample with methanol such that it is less than the maximum cholesterol concentration on the standard curve, based on the initial cholesterol content in the culture medium. Take 0.4 mL of the diluted sample, add 0.2 mL of ortho-phthalaldehyde, and then add 4 mL of mixed acid. Mix well, allow to stand for 20 minutes, and measure the absorbance at 550 nm.
2. BSH Plate Inoculation Experiment (Bile Salt Hydrolase Detection)
Materials and Concentrations
Test bacterial culture
MRS detection culture medium
Sterile filter paper
Procedure
- Place several pieces of sterile filter paper on the MRS detection culture medium. Inoculate 10 μL of bacterial culture on one filter paper piece. After inoculation, culture for 24 hours and record the formation of a precipitate ring.
3. GC Method for Short-Chain Fatty Acid Detection
Materials, Concentrations, and Instrument
- Test bacterial culture
- Acetic acid, propionic acid, butyric acid, and 2-ethylbutyric acid, all of chromatographic grade.
- Shimadzu GC-2010 Plus Gas Chromatograph equipped with a Flame Ionization Detector (FID) and an Rtx-Wax capillary column (30m×0.25mm, 0.25μm).
- Standard solution concentrations: Acetic acid 4.16 mmol/L, propionic acid 3.37 mmol/L, butyric acid 2.84 mmol/L.
- Internal standard: 2-ethylbutyric acid 2.21 mmol/L
Procedure
- GC analysis conditions: Utilize a DB-FFAP 122-3232 capillary column (30m×0.25mm, 0.25μm), an AOC 20i auto-sampler, and an FID detector. Hydrogen, nitrogen, and air pressures are set at 0.4 MPa. Temperature program: Initial temperature at 100°C, ramped up to 180°C at a rate of 10°C/min, held for 20 min, then ramped up to 230°C at a rate of 20°C/min, and held for 20 min, for a total analysis time of 18 min. The injection port temperature is set at 230°C, and the detector temperature is 250°C, with a split ratio of 1:10.
- Sample preparation: Take 500 μL of the bacterial culture, centrifuge at 4°C for 2 minutes at 10,000g. Take the supernatant and filter it through a 0.22 μm membrane, obtaining the sample solution.
- Detection: Take 40 μL of the sample solution, add 10 μL of 2-ethylbutyric acid, mix well, and inject 1 μL for analysis. (Before analyzing the samples, perform standard sample preparation with the same procedures to match the 2-ethylbutyric acid concentration with that of the sample solution.)
4. Enzyme Assay for Gene Expression
Cultivation and Evaluation of Fluorescence and Absorbance
Inoculate strains containing the mRFP reporter gene into 24-well plates with the required culture medium. After culturing all bacteria for 12-16 hours, use a plate reader (Thermo Fisher Scientific, USA) to measure fluorescence and absorbance to assess gene expression.
5. UHPLC for Cholesterol and Downstream Product Detection
Detection Conditions:
(1) HPLC Conditions:
- Column: Agilent InfinityLab Poroshell 120 EC-C18 3.0 x 100 mm, 2.7 μm (p/n 695975-302) or Agilent InfinityLab Poroshell 120 SB-C18 3.0 x 100 mm, 2.7 μm (p/n 685975-302) or Agilent InfinityLab Poroshell 120 EC-C18 3.0 x 100 mm, 1.9 μm (p/n 695675-302).
- Mobile Phase A: Water
- Mobile Phase B: Methanol
- Gradient: 0 to 10 minutes: 92 to 96% B, 10 to 12 minutes: 96% B, 12 to 16 minutes: 96 to 100% B, Stop time: 20 minutes, Post time: 2 minutes
- Flow Rate: 0.60 mL/min
- Column Temperature: 15°C
- Injection Volume: 20 μL
(2) MS Conditions:
- Ion Mode: APCI, Positive
- Drying Gas Temperature: 325°C
- Vaporizer: 350°C
- Drying Gas Flow: 4 L/min
- Nebulizer Pressure: 30 psi
- Capillary Voltage (+): 2000 V
- MRM Condition: AEMV, 500
6. BSH Enzyme Activity Assay
Add 100 μL of the enzymatic solution to 100 μL of 0.1 M phosphate buffer solution (pH 6.0), 20 μL of 200 mmol/L conjugated bile salt (TDCA), and 10 μL of paraffin oil. Mix and incubate at 37°C for 30 minutes (use samples with added conjugated bile salt as a control). Immediately add an equal volume of 0.15 mg/mL trichloroacetic acid to stop the reaction, mix well, centrifuge at 4°C, 8000 g for 15 minutes, and collect the supernatant.
Mix 300 μL of the supernatant (sample or control) with 1.7 mL of 0.2 g/L xanthydrol color reagent, shake well, heat in a boiling water bath for 14 minutes, cool in tap water for 3 minutes, and then measure the absorbance at a wavelength of 570 nm. Prepare a standard curve using glycine concentration (μmol/mL) as the x-axis and absorbance (A570nm) as the y-axis. Enzyme activity is defined as the amount of material that produces amino acids through the hydrolysis of conjugated bile salts by crude enzyme per unit time and per unit volume, expressed in μmol/(min·mL).
7. Protein Quantification
Standard Curve Preparation:
Prepare gradient standard solutions of bovine serum albumin (BSA) at various concentrations. Take 100 μL of the protein solution, add 1 mL of Bradford working solution, mix well, and measure the absorbance. Create a standard curve using protein concentration as the x-axis and absorbance as the y-axis.
Sample Testing:
Take the enzymatic solution and dilute it with 0.1 M phosphate buffer solution (pH 7.2) at various dilutions (2, 4, 6, 8, 16-fold). Take 100 μL of the above dilution, add 1 mL of the Bradford working solution, mix well, and measure the absorbance at 595 nm on a visible light spectrophotometer.
8. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis)
(1) Gel Preparation:
- Prepare 2 mL of 5% SDS-PAGE concentrated gel: 0.33 mL of 30% acrylamide, 0.25 mL of 1.0 M Tris-HCl (pH 6.8), 0.02 mL of 10% SDS, 0.02 mL of 10% ammonium persulfate, 0.001 mL of TEMED, and 1.4 mL of ddH2O.
- Prepare 5 mL of 12% SDS-PAGE separating gel: 2.0 mL of 30% acrylamide, 1.3 mL of 1.5 M Tris-HCl (pH 8.8), 0.05 mL of 10% SDS, 0.05 mL of 10% ammonium persulfate, 0.002 mL of TEMED, and 1.9 mL of ddH2O.
(2) Electrophoresis:
- Take 20 μL of protein extract, add 5 μL of loading buffer, boil in a water bath for 10 minutes, and briefly centrifuge for 6-7 seconds.
- Start loading samples from the second lane of the gel, use a 5 μL marker, and load 20 μL of protein extract.
- Run the gel at 80 V for 1.5 hours.
- After electrophoresis, wash the gel with deionized water 3-5 times to remove SDS and buffer. Then, stain the gel in Coomassie blue staining solution, incubate at room temperature on a shaker for 1 hour. Subsequently, destain in water for 2-3 days.
VI. Functional Verification
1. Pre-Cultivation
- Activate strains containing the IsmA gene.
- Cultivate IsmA gene-engineered bacteria in a culture medium containing 0.2 g/L cholesterol.
- Prepare egg yolk culture medium.
- Cultivate strains containing the BSH gene in MRS medium.
- Cultivate strains containing the BCoAT gene in LB medium.
- Cultivate strains containing the pSB1T3-FadR-FadB genes.
2. Functional Testing
- Test cholesterol content in cultures containing the IsmA gene.
- Use UHPLC to detect the IsmA crude enzyme.
- Test egg yolk cultures.
- Detect BSH enzyme activity on plates.
- Use GC to detect short-chain fatty acids.
- Use a plate reader to measure the expression effects.
V. Expression Analysis
1. BSH Enzyme Activity Testing Prepare crude enzyme extract by inoculating 100 μL of bacterial culture in 100 mL of LB medium, then centrifuge at 8,000 g for 3 minutes at 20°C. Collect the cell pellet and resuspend it in 0.1 M phosphate buffer solution (pH 6.0). Adjust the cell concentration to an absorbance of approximately 1.0 at 600 nm. Add 10 mmol/L dithiothreitol to reduce BSH oxidation, and then use sonication to break the cells for 30 minutes in an ice bath. Centrifuge the lysate at 10,000 g for 30 minutes at 4°C to remove cell debris, obtaining a cell-free extract. Use this extract to test BSH enzyme activity.
2. Protein Quantification Prepare a crude enzyme extract by inoculating 100 μL of bacterial culture in 100 mL of LB medium. After incubating at 37°C for 24 hours, centrifuge at 8,000 g for 3 minutes at 20°C. Collect the cell pellet and resuspend it in 0.1 M phosphate buffer solution (pH 7.2). Adjust the cell concentration to an absorbance of approximately 1.0 at 600 nm. Sonicate the cell suspension for 30 minutes in an ice bath. Centrifuge the lysate at 10,000 g for 30 minutes at 4°C to remove cell debris, obtaining a cell-free extract. Use this crude enzyme extract for protein quantification.
3. IsmA and BSH SDS-PAGE Prepare a crude enzyme extract by inoculating 100 μL of bacterial culture in 100 mL of LB medium. After incubating at 37°C for 24 hours, centrifuge at 8,000 g for 3 minutes at 20°C. Collect the cell pellet and resuspend it in 0.1 M phosphate buffer solution (pH 7.2). Adjust the cell concentration to an absorbance of approximately 1.0 at 600 nm. Sonicate the cell suspension for 30 minutes in an ice bath. Centrifuge the lysate at 10,000 g for 30 minutes at 4°C to remove cell debris, obtaining a cell-free extract. Use this extract for SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).