Experimental material
* Receptor Strain (EcN)
* LB medium
* 0.1M calcium chloride
preserve
* 30% glycerin
Experimental procedure:
The strain was activated by overnight culture
100ul of overnight activated bacterial solution was transferred to 100 mL of liquid LB and cultured for 2 h until OD was 0.5-0.6. Then pack the bacterial solution into 50 mL centrifuge tube (25 mL per tube) and precool the centrifuge at 4℃.
Centrifuge at 4000g for 10 minutes, discard the supernatant and add 20 mL0.1M CaCl2(5mL per tube) to the ice bath for 30 minutes per 100ml of the initial bacterial solution
Centrifuge, discard supernatant, add 1mL 15% 0.1M CaCl per tube (add 4 ml per 100 ml initial bacterial solution). Pack on ice into 1.5mL centrifuge tubes, 100 microliters each. For the preservation of receptive cells, 1ml of the mixture of calcium chloride and 30% glycerol (1:1) should be added and then subpackaged, and finally stored in the -80C refrigerator.

Experimental material and concentration
* Sample solution
*0.1mg/ml cholesterol working fluid
* Mixed acid (concentrated sulfuric acid: glacial acetic acid =1:1)
* Methanol
Experimental procedure:
First, the standard curve was obtained: 9 gradients of 0ml-0.4ml cholesterol working solution were successively taken, and 4ml mixed acid and 0.2ml phthalaldehyde were added to each gradient, and the volume of each gradient was supplemented to 4.6ml with methanol. The order of adding sample is working liquid, methanol, phthalaldehyde, mixed acid.
After adding the sample, mix it well, leave it for 20min, and measure the light absorption value at 550nm.
Sample testing: Use methanol to dilute the sample, based on the initial cholesterol content of the test medium, so that it is less than the maximum cholesterol concentration of the standard curve.
Take 0.4ml of diluted sample, add 0.2ml of phthalic aldehyde, then add 4ml of mixed acid, mix evenly, stand for 20min, and measure the absorption value at 550nm.

Experimental material and concentration * Bacterial solution to be tested *MRS Test medium * Sterile filter paper Experimental procedure: Several pieces of sterile filter paper were placed on MRS Test medium, and 10ul bacterial solution was inoculated on one filter paper. After inoculation, culture was performed for 24h and precipitation ring formation was recorded.

Experimental materials and concentrations and instruments
* Bacterial solution to be tested
* Acetic acid, propionic acid, butyric acid and 2-ethylbutyric acid are chromatographically pure.
* Shimadzu GC-2010 Plus Gas chromatograph with hydrogen Flame ionization detector (FID); Rtx-Wax elastic quartz capillary column (30m×0.25mm, 0.25μm).
* Standard concentration: 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
Experimental procedure:
Detection conditions: DB-FFAP 122-3232 elastic quartz capillary column (30m×0.25mm, 0.25μm) was selected for GC analysis. AOC 20i automatic sampler with FID detector. The pressure of hydrogen, nitrogen, and air is 0.4MPa. Programmed temperature setting: Initial temperature 100°C, temperature rise to 180°C at 10°C /min, hold for 20min, temperature rise to 230°C at 20°C /min, the total analysis time is 18min. The inlet temperature is 230°C; Detector temperature 250°C, shunt ratio: 1:10.
Sample preparation
Take 500ul bacterial solution and centrifuge at 4 ° C at 10000 g for 2min
Sample solution was obtained by taking the supernatant and filtering with 0.22um filter membrane
Detection
Take 40ul of sample solution, add 10ul of 2-ethylbutyric acid, mix well, and take 1ul of sample for testing. (Treatment of the standard before sample testing (operation with the sample solution, so that the concentration of 2-ethylbutyric acid is consistent with the sample solution) and injection)

Strains containing the mRFP reporter gene were inoculated into 24-well plates of the required medium. After 12-16 hours of culture, fluorescence and absorbance were measured by Thermo Fisher Scientific (USA) to test the expression function.

The strains containing IsmA gene were activated first, and then transferred to BCM medium (5ml) with 0.2g/l cholesterol concentration for 48h to prepare for cholesterol content change detection.
Strains containing BSH gene were inoculated in MRS Medium for 12h-24h to prepare for BSH plate inoculation experiment.
The strains containing BSH gene were activated first, and then transferred to M9 medium (5ml) containing 0.02g/l cholesterol and 0.1g/l bile salt, the only carbon source of deglucosed cholesterol, for 48h to prepare for cholesterol content change detection.
Strains containing BCoAT gene were inoculated in LB medium for 24h to prepare for detection of short-chain fatty acids by GC.
The affected strains containing pSB1T3-FadR-FadB gene were inoculated into 24-well plates supplemented with oleic acid-M9 medium and basic M9 medium, and all the bacteria were cultured aerobic and anaerobic for 12-16 hours, respectively, to prepare the gene expression degree detection by enzyme labeler.

Strains containing IsmA gene or BSH gene for cholesterol detection were cultured in the corresponding medium for 48h, diluted with 5ml methanol in the bacterial solution, shaken well, and sampled to detect cholesterol content by OPA method. The culture medium without added bacteria was used as blank control, and EcN bacteria and empty plasmid transforming bacteria were used as Yin and Yang control.
After the preparation of BSH plate inoculation experiment was completed, BSH qualitative plate inoculation experiment was carried out. EcN bacteria were compared with empty plasmid bacteria.
After the strains containing GC gene were cultured, GC detection was performed to detect the content of short-chain fatty acids.