Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain
Experimental procedure:
1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack, Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.
2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2 boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it was cleaned up properly afterwards.
3. Activated strains: 1 tube of Escherichia coli DH5α/ empty vector 1 and 1 tube of DH5α/ carrying MFS gene. One tube of Escherichia coli DH5α/ empty vector 2, one tube of DH5α/ carrying ismA gene, and one tube of DH5α/ carrying MFS gene
galU gene, turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the tube orifice and tube stopper, put 100 μL preservation bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the tube orifice and tube stopper again. The test tube was sealed, bundled, and incubated on a shaker overnight (150 rpm, 37℃).
 

Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR
Experimental procedure:
1. Plasmid extraction: Four kinds of plasmids DH5α/ empty vector 1,2,MFS,ismA,galU were extracted according to the instruction of plasmid extraction kit, and the bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added and resuspended, p2 was mixed upside down, p3 was mixed upside down, and then centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.

2. Diluted primer
The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water was added to dilute the primer to the appropriate concentration
3.PCR reaction:
(1) Target genes pheP, PAL and two kinds of vector DNA fragments were amplified by high fidelity enzyme Pfu. The reaction system is as follows:
Pfu 0.5 μL
10xPfu buffer 5 μL
Primer 1 1 μL
Primer 2 1 μL
Template (plasmid extracted above) : 1μL
25μL dNTP Mixture
20 μL of sterilized water
(2) Perform the PCR amplification procedure
At 98 ° C for 5 min
The following 3-step reaction was performed for 30 cycles
98℃ 10s
55℃ 5s 
72℃ 1min/kb
72℃ 8 min
The plasmids, primers and products required for PCR are shown in the table below:
    

Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental procedure:
1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled.

2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit
Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min, and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA concentration was determined using Nanodrop.

3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:
1 μg of DNA
1 μL of EcoRI
1 μL XhoI
4 μL 10xY buffer
H2O up to 20μL
The enzyme was digested at 37 ° C for 4h
4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.

Experimental plan: Activation of E. coli DH5α and Rosetta to prepare competent cells
Experimental procedure:
1. Activated strains: including Escherichia coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper, connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube stopper again. After sealing the tube, the tube was bound and incubated on a shaker overnight (220 rpm, 37℃). The OD value of the bacteria solution was measured, and the competent state was prepared when the OD value was about 0.5.

 
2. Preparation of competent cells:
(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a refrigerator at 4 ° C.
(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard, and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again (4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.

Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform ligation products
Experimental procedure:
1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).

2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment volume ratio can also be calculated according to the instructions) :
T4 DNA ligase 1 μL
2 μL of 10 x ligation buffer
	1 μL of Linear plasmid
	Insert 6 μL of DNA
	Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was stored at -20 ° C.
3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1 mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100 μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.

Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation
Experimental procedure:
1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as templates for colony PCR using Taq enzyme. The primers are TF, TR. The reaction system and procedures were carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed

2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp antibiotic was added to expand the culture.
3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.
4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.

Various concentrations of oleic acid were added to LB medium and E.coli Rosetta was inoculated, and the OD600 values of E.coli Rosetta were measured using a spectrophotometer. The results showed that 5 mM oleic acid almost did not affect the growth of bacteria, while 10 mM oleic acid inhibited the growth of bacteria.

pOleic acid-mRFP was constructed in pSB1A3. The recombinant plasmid was transformed into E.coli Rosetta last. Various concentrations of oleic acid were added to LB medium and E.coli Rosetta/pOleic acid-mrFP was inoculated. Adjust the initial OD600 to 0.1. After 12 hours, OD600 values and mRFP fluorescence intensity were measured using a microplate reader. The results showed that the oleic acid promoter induced about 4.5-fold change in the engineered bacteria under the induction of 0mM and 5 mM oleic acid.

Each bacterial culture was grown in LB medium as previously described and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanolcholesterol stock solution was added to LB medium to achieve a final concentration of 80 μg/mL. Cholesterol quantification kit (mlbio, ml094955) was used to measure the cholesterol concentration in each medium. OD600 and cholesterol concentrations were measured again after 12 h of culture. To normalize the data, we calculated Δcholesterol/ΔOD600, which represents the uptake of cholesterol by bacteria during growth. All values were determined based on the mean of all repeated measurements.

The ismA gene was cloned into the pET28a plasmid, validated and transformed into Escherichia coli Rosetta. The recombinant strain Rosetta was then incubated in LB medium containing 0.5 mM IPTG for 12 h at 16˚C. Ten mL of the bacterial culture was collected, the medium supernatant was discarded by centrifugation, the cell pellet was resuspended in 20 mM Tris-HCl (pH 7.0), the crude enzyme solution was collected by pre-sonication on ice, and the protein concentration was determined by the Bradford method. The 5 mg/mL crude enzyme solution was incubated with 200 μM cholesterol for 12 h. Cholesterol concentration was subsequently determined based on fluorescence emission using the Amplex™Red Cholesterol Assay Kit (Sigma, A12216).

ismA expression was controlled by the oleic acid promoter at week 12
The ismA gene was cloned downstream of pOleic acid and validated before transformation into Escherichia coli Rosetta. The recombinant strain Rosetta was then placed in LB medium containing various concentrations of oleic acid. After 12 hours, the crude enzyme was extracted as described previously. And 5 mg/mL of the crude enzyme solution was incubated with 200 μM cholesterol for 12 h. Cholesterol concentration was subsequently determined according to fluorescence emission using the Amplex™Red Cholesterol Assay Kit (Sigma, A12216).

Overexpression of galU enhanced EPS production in E. coli Rosetta.
Each bacterial culture was grown in LB medium as previously described and then diluted to OD600 = 0.1. At 0 h, 8 mg/mL ethanolcholesterol stock solution was added to LB medium to achieve a final concentration of 80 μg/mL. Cholesterol quantification kit (mlbio, ml094955) was used to measure the cholesterol concentration in each medium. OD600 and cholesterol concentrations were measured again after 12 h of culture. Δcholesterol/ΔOD600 was calculated, which indicates how well the bacteria adsorb cholesterol during growth