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 E. coli DH5α/ empty vector 1 and 1 tube of DH5α/ pheP gene. One tube of E. coli DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube orifice and plug, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the test tube orifice and plug again. After sealing the tube, the tube was 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 and one tube DH5α/ pheP gene were extracted according to the instruction of plasmid extraction kit. One tube of E. coli DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. The bacterial solution was centrifuged for 1min and then the precipitate was removed. 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.

Strains and plasmid sample white floc after drip into the P3
 

The experimental work
2. Dilute the primers 
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

Centrifuge dry powder primer manipulation

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.

Plot of gel electrophoresis results
2. Gel recovery: The PCR product DNA gel was recovered according to the gel recovery kit instructions
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.

Completed rubber recycling product concentration determination of DNA
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.

The researchers cut gel

Photo of the experimenter at work

Experimental plan: E. coli DH5α and Rosetta were activated 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. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).
Experimental procedure:
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 packed 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℃.

1. Pre-activated aliquots of DH5α and Rosetta bacterial samples
2. Bacterial samples in an ice bath
3 Add the CaCl2 solution
4. After secondary centrifugation of the bacterial sample, white precipitate was produced
5 Add glycerol CaCl2 mixing buffer

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).

Pour the liquid LB medium into the disposable Petri dish
2 Ligation reaction: The vector was ligated by double enzyme digestion of the target fragment. 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
	Connections were made using a PCR apparatus at 16 ° C for 1h. 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.

Experimental plan:
Experimental procedure:
1 Incubation, ELISA: To measure the capacity of whole cells to metabolization of phe, the PAL engineered strain was resuspended to an OD600 of 0.1 in 1 mL of Phe assay buffer (M9 0.5% glucose containing 1 mM millimoles per liter of Phe) and incubated in microtubes. The amount of Phe was then determined according to the phenylalanine ELISA kit. , the results are shown in FIG. A below.
2. The strains were resuspended in 1 mL Phe experiment buffer (M9 0.5% glucose containing 1 mM Phe) to an OD600 of 0.1, placed in microtubes, and incubated at 37°C for 1h. TCA concentration was determined with OD290 as shown in FIG. B.
3 Simultaneously determine the effect of pH on PAL enzyme activity, as shown in Figure C.

Experimental plan: To test the effect of the hypoxic promoter and PAL expression
Experimental procedure:
1. The tested engineering bacteria were statically cultured in a CO2 incubator (adjust the O2 concentration to 0%, 10% and 20%). After 16h of growth, the absorbance (OD600) and mRFP fluorescence intensity data were measured using a microplate reader.
2. The tested engineered bacteria were statically cultured in a CO2 incubator (O2 concentration was adjusted to 0%, 10% and 20%). The strains were resuspended in 1 mL Phe experiment buffer (M9 0.5% glucose containing 1 mM Phe) to an OD600 of 0.1, placed in microtubes, and incubated at 37°C for 1h. TCA concentration was determined using OD290. The Phe content was measured using an Elisa kit.

Experimental plan: Promoter testing and determination of PAL activity
Experimental procedure:
1 The strain was resuspended in 1 mM Phe experiment buffer to an OD600 of 0.1 and placed in a microtube, and an additional 1 mM TCA was added to the test group. The cells were incubated at 37°C for 1h. Phe content was determined according to the phenylalanine ELISA kit, and TCA content was tested using OD290.

Experimental plan: The signal amplification switch of TP901 was coupled to test the fluorescent protein expression
Experimental procedure:
1. The same procedure as above was used to construct the plasmid, mRFP gene was coupled to the downstream of TCA promoter pSenCA, and the fluorescent protein expression was measured under different TCA content.
2. Fluorescence microscope verification experiment:
(1) Sample preparation: appropriate amount (about 30 μl) of wild type Rosetta and Rosetta-engineered bacteria droplets were dropped onto the slide and covered with a cover slip.
(2) Turn on the switch of the power control box of the high-pressure mercury lamp.
(3) Insert the light stopper to interrupt the light path.
(4) Preheat for 5-10 min.
(5) Place the slide with the sample on the stage.
(6) Select the objective lens (in order of first low power, then high power).
(7) Rotate the turntable of the spectroscope assembly to select the spectroscope assembly for observing mRFP (red fluorescence).
(8) Adjust the focal length through thick and thin screws.
(9) Open the computer connected to the microscope and click on the digital imaging system software to collect digital images.
3. Microplate reader was used to test fluorescent protein expression: 100 μL of wild-type Rosetta, recombinant strain solution was taken in an ultra-clean stage. The supernatant was added to a 96-well plate for 4-6 replicates. The detection conditions of the microplate reader were set (mRFP excitation λ: 584 nm/10 nm, emission λ: 607/10 nm), and the readings were taken. The data were recorded using Excel.