Preparing Luria - Bertani (LB) medium
Goal: Used to cultivate E. coli

Apparatus	Amount
Sterilised duran bottle, cup	2
Autoclave	1
Electronic Balance	1

Liquid Luria - Bertani a (Liquid LB) materials

Reagents	Volume/Mass
Tryptone	10g
Yeast extract	5g
Sodium chloride (NaCl)	10g
Double Distilled water (ddH2O)	1000 ml

Procedures:
✓ To prepare 1 liter of liquid LB medium, add 10g of tryptone, 5g of yeast extract, and 10g of NaCl to 950 ml of deionized water.
✓ Stir the container until the solutes dissolve, and adjust the pH to 7.0 using 5 M NaOH. Fill up to 1 liter with deionized water.
✓ Sterilize by steam for 15 minutes at 121℃.
Solid Luria - Bertani (Solid LB) materials

Reagent	Volume/Mass
Liquid LB	100 ml
Agar	1.5g

Procedures:
✓ First, add 1.5 g of agar powder to 100 ml of liquid LB medium to prepare the solid LB medium.
✓ Place the molten LB agar medium in a 55°C water bath after high-pressure sterilization.
✓ When the temperature drops to 55°C, add the antibiotics to prevent deactivation due to high temperature, and mix thoroughly.
✓ Pour 10 ml of the medium onto each plate. After pouring the mixed medium into Petri dishes, open the lids and expose them to UV light for 10-15 minutes, sealing the edges with sealing tape.

Goal: Remove RNA to separate plasmid DNA from bacterial genomic DNA, then remove proteins and other impurities to obtain relatively pure plasmid DNA.

Apparatus	Amount
Centrifuge	1
Spin column	4
Thermostat	1

Reagents	Volume/Mass
Solution I: Bacterial Suspension	250 ul/time
Solution II: Cell Lysis Buffer	250 ul/time
Solution III: Neutralization Buffer	350 ul/time
Wash solution	750 ul/time

Procedures:
1. Centrifugation and Pellet Preparation
Centrifuge 1-5 mL of bacterial culture (P1, P2, Azurin, anti-PD-L1) at 12,000 rpm for 1 minute and discard the supernatant.
2. Resuspension
Add 250 µL of Solution I to the pellet in the centrifuge tube and use a vortex mixer to resuspend the bacterial pellet thoroughly.
3. Lysis
Add 250 µL of Solution II to the tube and gently invert 6-8 times to fully lyse the bacteria; the solution should become clear and viscous.
4. Precipitation
Add 350 µL of Solution III to the tube, invert 6-8 times to mix thoroughly, and a white precipitate should form.
Centrifuge at 12,000 rpm for 10 minutes and transfer the supernatant to a clean centrifuge tube.
5. Precipitation with Ethanol
Add 0.35 volumes of absolute ethanol to the supernatant and mix thoroughly.
6. Column Loading and Initial Centrifugation
Transfer the supernatant to a column, allow it to sit at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, discard the waste in the collection tube, and place the column back into the collection tube.
7. Wash Steps
Add 750 µL of wash buffer to the column, centrifuge at 12,000 rpm for 1 minute, and place the column back into the collection tube.
Add 700 µL of wash buffer to the column, centrifuge at 12,000 rpm for 1 minute, and place the column back into the collection tube.
Centrifuge at 12,000 rpm for 2 minutes, then place the column open side up at room temperature or in a 50°C incubator for several minutes.
8. Elution
Place the column into a clean centrifuge tube, and add 50-200 µL of pre-warmed elution buffer (65°C) to the center of the membrane; allow it to sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute.
Repeat the elution by adding the collected elution buffer back to the column, allowing it to sit at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.

Goal： In vitro, DNA amplification technology can rapidly and specifically amplify any target DNA outside of a living organism.

Apparatus	Amount
Pipette (range: 2-20ul)	some
Pipette (range: 20ul-100ul)	some
Centrifuge	1
PCR amplification instrument	1

Reagents	Volume/Mass
Target gene (Plactate1-Azurin; Plactate1-anti-PD-L1; Plactate2-Azurin; Plactate2-anti-PD-L1)	2 ul
2*Mix (dNTPs, DNA polymerase, Buffer, Magnesium ions)	25 ul
Primers (F/R)	F: 2 ul R: 2 ul
ddH2O	19 ul

Procedures:
✓ Add different target genes, mix solution, primers, and double distilled water into each tube, and prepare the initial solutions.
✓ Setup the PCR program

Progress	Temperature	Time
Initial Denaturation	95 ℃	5 min
Denaturation	95 ℃	30s	repeat 30 times
Annealing	55 ℃	30s
Extension	72 ℃	1kb/min
Final Extension	72 ℃	10 min
Hold	4 ℃	∞

Goal: Separation and Identification of DNA and RNA Molecular Mixtures

Apparatus	Amount
Pipette (range: 2ul-20ul)	some
Pipette (range: 20ul-200ul)	some
Weighing paper	1 piece
Electronic balance	1
Microwave oven	1
Spatula	1
Graduated cylinder	1
Comb	2
Electrophoresis chamber	1

Reagents	Volume/Mass
Agarose	5g
Tris Acetate-EDTA buffer (TAE)	50 ml
Red dye	3ul
Marker	5ul

Procedures:
1.Preparing the gel
✓ Place the weighing paper on the electronic balance and weigh 5g of agarose. Transfer it to a conical flask.
✓ Add 50 ml of TAE buffer to the conical flask and mix.
✓ Microwave it for 3 minutes until the agarose granules dissolve completely.
✓ Use a pipette (2μL-20μL range) to add 5 μL of nucleic acid dye to the flask.
✓ Pour the mixture into the mold and insert a comb.
✓ Wait 10 minutes for the gel to solidify.
2.Electrophoresis
✓ Install the electrophoresis chamber.
✓ Pour the cooled gel gently onto the horizontal plate of the electrophoresis chamber.
✓ After it solidifies, add electrophoresis buffer into the chamber and remove the comb.
✓ Add the sample mixture to the wells.
✓ Connect one end of the electrophoresis wells to the negative electrode and the other end to the positive electrode. Turn on the power, set the voltage to 180V, and run electrophoresis for 20 minutes.
✓ Remove the gel and observe under UV light.
✓ DNA bands will appear as red fluorescent bands.

Goal: Extract pure target gene bands and apply them in forward experiments

Apparatus	Amount
Scalpel	1
UV transilluminator	1

Reagents	Volume/Mass
Sol liquid	300ul/0.1g DNA gel block
Rinse solution	600ul/time

Procedures:
1. After agar-gel electrophoresis, a single DNA strip of interest is cut from the agar-gel and weighted
2. Add 600μL sol solution to the gel block and place in a water bath at 55℃ for 10 min. During the process, gently turn the centrifuge tube up and down continuously to ensure that the glue block is fully dissolved
3. The solution was added to an adsorption column and centrifuged at 12000pm for 60 seconds
4. Add 600μL bleaching solution to the adsorption column and centrifuge at 12000rpm for 1min, discard the waste liquid, and put the adsorption column into the collection tube
5. Add 600μL bleach solution into the adsorption column, centrifuge 12000rpm for 1min, discard the waste liquid, and put the adsorption column into the collection tube
6. Centrifuge at 12000rpm for 2min, remove the bleach solution as much as possible, and place the adsorption column open at room temperature or 50℃ in a temperature box
7. Place the adsorption column into a clean centrifuge tube, drop the appropriate amount of eluent preheated in a water bath at 65℃ into the center of the adsorption film, place at room temperature for 2min, and centrifuge at 12000rpm for l min
8. DNA products stored at -20℃

Goal: Achieve the recombinant plasmids

Apparatus	Amount
Pipettes (range: 2-20ul/20-200ul)	4
Water bath	1

Reagents	Volume/Mass
Linearized plasmid	2μL
Target gene	1μL
2 × ClonExpress Mix	5μL
ddH2O	2μL

Procedures:
Prepare the following reaction system on the ice. Use a pipette to gently suck and mix (do not shake and mix), and briefly centrifuge to collect the reaction liquid to the bottom of the tube. 50℃, 15 min; Reduce to 4 ° C or cool immediately on ice

Procedures:
1. Inoculating ECN 1917 and preparing competent ECN 1917 cells
Inoculate overnight ECN 1917 culture with 1% volume into fresh medium and grow at 37°C, 220 rpm until OD600 reaches 0.3 (2.5 hours).
Harvest the bacteria by chilling on ice for 5 minutes, then centrifuge at 4°C, 5000 rpm for 5 minutes.
Wash the pellet with cold deionised water and centrifuge at 4°C, 5000 rpm for 5 minutes.
Resuspend the pellet in 2 ml of cold 0.05 M CaCl2 and 15% glycerol solution, gently mix, and chill on ice for 5 minutes.
Add another 2 ml of cold 0.05 M CaCl2 and 15% glycerol solution and gently mix to obtain competent cell suspension. Aliquot into 50-100 µl portions and store at -70°C.

2. Heat recovery
Heat the sample at 42°C for 45 seconds, then immediately place it on ice to cool for 2-3 minutes.

3. Recovery
Add 900 μL of LB medium (without antibiotics) to each tube.
Gently mix the contents and incubate the tubes in a shaking incubator at 37°C and 200 rpm for 45-60 minutes to allow the cells to recover and express the plasmid-encoded genes.

4. Plating
Centrifuge at 5000 rpm (2,500 × g) for 5 min and discard 900 μL supernatant. The bacteria sediment was suspended with the remaining medium and lightly coated with a sterile coating stick on a plate containing Amp resistance.
Then, invert the plates and incubate at 37°C for 12-16 hours to allow colonies to grow.

PCR program

Apparatus	Amount
Pipette (range: 2-20ul)	1
Pipette (range: 20ul-100ul)	1
Centrifuge	1
PCR amplification instrument	1

Reagents	Volume/Mass
Target gene (Plactate1-Azurin; Plactate1-anti-PD-L1; Plactate2-Azurin; Plactate2-anti-PD-L1; DNA polymerase; Magnesium ions)	2ul
2*Mix (dNTPs, DNA polymerase, Buffer, Magnesium ions)	10ul
Primers (F/R)	F: 1ul R: 1ul
ddH2O	8ul

Procedures:
✓ Add different target genes, mix solution, primers, and double distilled water into each tube, and prepare the initial solutions.
✓ Setup the PCR program

Progress	Temperature	Time
Initial Denaturation	95 ℃	5 min
Denaturation	95 ℃	30s
Annealing	55 ℃	30s (repeat 30 times)
Extension	72 ℃	1 min
Final Extension	72 ℃	10 min
Hold	4 ℃	∞

Gel Electrophoresis
Prepare the Agarose Gel:
✓ Gel Preparation: Dissolve agarose powder in a TAE buffer solution by heating. The agarose concentration typically ranges from 0.7% to 2.0%, depending on the size of the DNA fragments you want to resolve.
✓ Cooling and Casting: Allow the agarose solution to cool to about 50-60°C before pouring it into a gel casting tray. Insert a comb to create wells for loading the samples. Let the gel solidify at room temperature (30 minutes).
Prepare the Gel Electrophoresis Apparatus:
✓ Setup: Place the solidified agarose gel into the electrophoresis tank and cover it with a running buffer (TAE).
✓ Buffer Preparation: Prepare the electrophoresis buffer according to the manufacturer's instructions. This buffer facilitates the conduction of electricity through the gel and helps maintain the pH.
Load the Samples:
✓ Mix with Loading Dye: Mix the PCR products with a loading dye. This dye helps to visualize the sample and adds density so that the samples sink into the wells.
✓ Loading: Carefully pipette the PCR products and a DNA ladder into the gel wells.
Run the Gel:
✓ Electrophoresis: Apply an electric current across the gel. DNA fragments will migrate through the gel matrix towards the positive electrode (anode) because DNA is negatively charged. Smaller fragments move faster and travel further than larger ones.
Visualize the DNA:
✓ Staining: After electrophoresis, stain the gel with a DNA-binding dye. This dye intercalates with the DNA and fluoresces under UV light.
✓ Imaging: Use a gel documentation system or UV transilluminator to visualize and capture an image of the gel. Bands corresponding to DNA fragments will appear, allowing you to compare the size of the PCR products with the DNA ladder.

GFP Fluorescence Measurement
✓ Fluorescence Microscopy Detection: sfGFP fluorescence emission.
✓ Spectrophotometer Measurement:
Black 96-well plates, 470 nm excitation, 509 nm emission, and read the value;
Transparent 96-well plates, OD600;
Relative fluorescence intensity = read value of the former / read value of the latter.

Apparatus	Amounts
Gel Molds	2
Pipettes and pipette tips (2-20ul / 100-1000ul)	2
Magnetic Stirrer	1
Stirring Rod	1
Water bath machine	1

Reagents	Volume or Mass
Tris-HCl (1.5 M, pH 8.8)	2.5 mL (for a 12% gel)
Tris-HCl (0.5 M, pH 6.8)	1.25 mL (for a 12% gel)
ddH2O	Up to the top of the gel mold
Sample Buffer (different lactic acid concentrations of P1, P2, anti-PD L1, Azurin)	10 µL per sample
Electrophoresis Buffer	1 L (Tris-Glycine-SDS buffer)
Coomassie Brilliant Blue	5 µL

Procedures:
1. Prepare Equipment and Materials
✓ Gel electrophoresis equipment (electrophoresis tank, power supply)
✓ Gel components: acrylamide, crosslinker (N, N'-methylene bisacrylamide), buffer solution
✓ Sample buffer (P1, P2, Anti-PD L1, Azurin)
✓ Coomassie Brilliant Blue

2. Gel Preparation
2.1 Prepare Separating Gel
✓ Mix acrylamide solution: acrylamide, crosslinker, and buffer solution.
✓ Pour into gel mold: Usually, the separating gel is poured first and then allowed to solidify.
2.2 Prepare Stacking Gel
✓ Mix stacking gel solution: acrylamide, crosslinker, and buffer solution.
✓ Pour into gel mold on top of the solidified separating gel.
2.3Insert Comb
✓ Insert a comb to form wells in the gel, ensuring it solidifies with the wells intact.
2.4 Solidify Gel
✓ Allow the gel solution in the mold to solidify at room temperature or in a 4°C refrigerator, typically for 30 minutes to 1 hour.

3. Sample Preparation
3.1 Process Samples
✓ Mix samples with sample buffer, usually containing SDS and reducing agents.
✓ Heat samples (usually at 95°C for 5-10 minutes) to denature and linearize proteins.
3.2 Cool Samples
✓ Cool the samples to room temperature after heating and prepare for loading.

4. Electrophoresis Steps
4.1 Assemble Gel
✓ Remove the solidified gel from the mold and place it in the electrophoresis tank.
✓ Add electrophoresis buffer to ensure it covers the gel.
4.2 Load Samples
✓ Carefully load the prepared samples into the gel wells using a micropipette.
4.3 Run Electrophoresis
✓ Connect the electrophoresis tank to a power supply and set the appropriate voltage (120V).
✓ Run electrophoresis until the dye front reaches the bottom of the gel (usually 1 hour).

5. Post-Processing
5.1 Stain Gel
✓ Treat the gel with a staining reagent (Coomassie Brilliant Blue) to visualize protein bands.
5.2 Destain Gel
✓ After staining, place the gel in a destaining solution or water to remove background staining and reveal clear protein bands.
5.3 Analyze Results
✓ Observe and record the position and intensity of protein bands.
✓ Estimate protein molecular weights using molecular weight markers.