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, 1 tube of DH5α/ carrying try gene,1 tube of DH5α/ carrying gshF gene, and 1 tube of DH5α/ carrying GshF-pepG gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the orifice and plug of the test tube, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the orifice and plug of the test tube 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. Extraction of plasmids: four kinds of plasmids were extracted according to the instructions of the 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. 
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
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
5 μL of 10xPfu buffer
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: E. coli DH5α and Rosetta were activated to prepare competent cells
Experimental procedure:
1. Activated strains: including E. 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 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.

Experimental procedure:
Different concentrations of melanin were added to LB medium, and the absorbance at 400nm was measured and recorded. Establishing a standard curve
LB medium was supplemented with 50 mg/L L-DOPA and 8 mg/L L-tyrosine as substrates, and bacteria were grown at 37°C on a shaker (180rpm). Every 4 hours, 2mL of the bacterial solution was transferred to the EP tube, the cells were broken up and centrifuged, and the absorbance at 400nm was measured and recorded. The production curve was measured within 24h.

Experimental procedure:
Bacteria were grown at 37°C and 25°C on a shaker (180rpm). OD400 was measured at 24h and the melanin concentration was converted from a standard curve. The pH of LB medium was adjusted to 7,6,5 using hydrochloric acid. Bacteria were grown in a shaker (180rpm) at 37 ° C. OD400 was measured at 24h and the melanin concentration was converted from a standard curve.

Experimental procedure:
To measure GSH production, strains were resuspended in LB medium to an OD600 of 0.1 and incubated for 2h at 37°C. The GSH content was determined by the reduced glutathione (GSH) content detection kit. Initially, the strains were resuspended in LB medium to an OD600 of 0.1, incubated at 37°C, and sampled at 4, 6, 8, and 12 for OD600 measurement to determine the effect of GshF on bacterial growth. Wild type E. coli was resuspended in LB medium to OD600 of 0.1, incubated at 37°C, and different amounts of glutathione were added. Samples were taken at 4, 6, 8, and 12h to determine the effect of OD600 and glutathione on bacterial growth. The recombinant vector was transformed into E. coli BL21 cells, and positive clones were screened on LB AGAR plates containing ampicillin. The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The next day, 1 g of bacteria was collected by centrifugation, resuspended in PBS (pH 7.4), and cells were then lysed by sonication (150 W, 1s sonication, 3s interval, for a total of 20 min) to obtain cell lysates. The 10 mL PBS reaction mixture contained 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamate, 20 mM L-cysteine, and 20 mM glycine. After 1 h of reaction at 37°C, the amount of GSH was determined using a reduced glutathione (GSH) content assay kit. The tested engineered bacteria were statically cultured in a CO2 incubator (O2 concentration was adjusted to 0%, 20% and 30%). After incubation at 37°C for 2h. The content of GSH was determined by reduced glutathione (GSH) content detection kit.

Experimental procedure:
The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The bacterial precipitate was collected by centrifugation the next day, resuspended in 20 mM Tri-HCl buffer (containing 150 mM NaCl, pH 8.0), and then sonicated (150 W, 1s sonication, 3s interval, for a total of 20 min) to obtain the crude enzyme solution. The protein concentration of the crude enzyme solution was determined using the Bradford Assay kit (Solarbio, China). 100 μg of the crude enzyme solution was digested using 2U thrombin for 4h at 25 ° C. 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamic acid, 20 mM L-cysteine and 20 mM glycine were added to the cleavage product, and the reaction was carried out at 37 ° C for 1 hour. The GSH content was determined using the GSH content detection kit.