Goal: Create a nutritive environment for bacterial cultivation and microorganism activation.

Materials:

• Yeast Extract
• Sodium chloride
• dd H₂O
• Sterilized duran bottle, cap
• Lab Refrigerator
• Tryptone
• Autoclave
• Agar

Procedures:

1. Prepare LB Medium (for E. coli recombinant strain maintenance/cultivation)
2. Measure 20g premixed LB broth powder (Tryptone:Yeast extract:NaCl = 2:1:1)
3. Note: For solid medium, add 1.5% agar
4. Transfer powder to sterile Duran bottle
5. Add dd H₂O to final volume of 1000 mL
6. Secure cap and autoclave (≥20 min at 121°C)
7. Cool to room temperature

1. Construction of pRED
1. Obtaining the red light-induced system empty plasmid

Goal: To construct the red light-induced expression empty plasmid

Materials:

1) 2xPrimeStarMix

2) template

3) double distilled water (ddH₂O)

4) Primer-R

5) Primer-F

6) centrifuge tube

Procedures:

1) Combine 25µL of 2xPrimerStarMix, 1µL of template, 1µL of primer-R, and 1µL of primer-F in a centrifuge tube. Adjust the volume to 50µL with ddH₂O. Centrifuge the mixture briefly if bubbles are present.

2) Transfer the mixture to a PCR thermal cycler and denature the fragments at 95˚C for 3 min.

3) Run 32 cycles consisting of 30 seconds denaturation at 95˚C, 30 seconds annealing at 55˚C, and 2 min extension at 72˚C.

4) Perform a final extension step at 72˚C for 1 min.

5) Store the fragments at 4˚C if not used immediately.

2. Agarose gel construction, deployment and recycle

Goal: To produce a functional and well-shaped gel for electrophoresis

Materials:

1) Casting Tray

2) Well comb

3) Microwave

4) 1x TAE

5) Agarose M

6) 10000x Nucleic acid gel stain

7) Erlenmeyer flask

Procedures:

1. Dissolve 0.3g of agarose in a 30mL Erlenmeyer flask.
2. Add TAE (running buffer) to the flask.
3. Heat the mixture using a microwave.
4. Melt the mixture in 30-second intervals.
5. Check for the formation of bubbles to ensure thorough mixing.
6. Incorporate 3μl of nucleic acid gel stain (10000x).
7. Allow the mixture to cool for 5-10 mins.
8. Transfer the agarose mixture into the casting tray.
9. Position the appropriate well comb to create wells in the gel.
10. Allow the gel to solidify for approximately 30 mins.
11. Remove the comb and transfer the gel to the gel box.
3. Agarose gel electrophoresis

Goal: To verify the DNA bands’ lengths of the plasmid extracted and recycle later.

Procedures:

1. Take out 50µl from each sample and mix with 5µl of loading buffer (10x).
2. Prepare the horizontal electrophoresis system for gel placement.
3. Mix 50µl of the combined sample solution until uniform in color.
4. Load the marker in the first well followed by 50µl of each sample into the respective wells.
5. Run the procedure at 120V for 20 mins.
6. Remove the gel from the apparatus and proceed with the subsequent steps.
4. DNA Gel Extraction

Goal: To recover the target fragment in the agarose gel.

Materials:

1) Agarose gel with target fragment

2) 1.5 mL EP tube

3) Agarose Gel Electrophoresis Recovery Kit

Procedures:

1. Excise the gel slice containing the 5984 bp fragment, removing excess gel, and transfer it to a 1.5 mL EP tube.
2. Add 500 microliters of buffer B2 and incubate the tube in a 50°C water bath until the gel is completely dissolved.
3. Transfer the melted gel solution into a purification column placed in a collection tube and centrifuge at 8000 g for 30 seconds.
4. Discard the collection tube, reinsert the purification column, add 500 microliters of ethanol-containing wash solution, and centrifuge at 9000 g for 30 seconds. Empty the collection tube.
5. Repeat the wash step.
6. Centrifuge at 9000 g for 1 min, then briefly open the tube cap to allow ethanol evaporation for one min.
7. Transfer the purification column to a new 1.5 mL EP tube. Add 20 microliters of elution buffer to the center of the purification column.
8. Close the lid and incubate the tube for 1 min, followed by centrifugation at 9000 g for 1 min.
9. Discard the purification column and store the DNA in the EP tube at 4°C.
5. Homologous recombination to construct plasmid pRED

Goal: Constructing the target plasmid through homologous recombination.

Materials:

1) ClonExpress Mix (2x)

2) ddH2O

3) PCR products of target fragment

Procedures:

1) Quantify the concentration of the gel-recovered fragments.

2) According to the instructions, prepare the system.

3) Incubate at 50 ℃ for 15 mins.

6. Transferring the recombined fragment into DH5α

Goal: Transform the constructed plasmid into Escherichia coli for validation and amplification.

Materials:

1) Plasmid after ligation

2) E. coli in a competent state

Procedures:

1. Combine the ligated samples with E. coli samples.
2. Chill on ice for 20 mins.
3. Heat shock at 42 ℃ for 45 seconds, then promptly transfer to ice for 2-3 mins.
4. Mix with 900μL LB Agar solution and incubate for 30 mins.
5. Centrifuge at 5000xg for 3 mins, remove and discard 900μL of supernatant.
6. Apply the final product to agar plates and culture for 12-16 hours at 37 degrees Celsius.
7. Colony PCR Validation

Goal: Verify the transformants and inoculate them to grow with the target gRNA.

Materials:

1) Taq Master Mix (2x)

2) ddH2O

3) Culture plates post-transformation

Procedures:

1. Set up the system (20 μL total volume: 10 μL ClonExpress Mix, 1 μL primer-F, 1 μL primer-R, 8 μL ddH2O).
2. Using a sterile 2.5 μL pipette tip, pick a single clone and transfer it into the system.
3. Place the mixture into a PCR thermal cycler, following the PCR program according to the manufacturer's instructions.
4. Verify the successful construction of plasmid by performing agarose gel electrophoresis, where the presence of the correct bands confirms the outcome.
8. Extracting the plasmid

Goal: To obtain the plasmid pRED from DH5α

Materials:

Plasmid Extraction Kit

Procedures:

1. In a 2 mL centrifuge tube, collect 2 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, and remove the supernatant carefully.
2. Repeat the centrifugation step with another 2 mL of bacterial culture, discarding the supernatant afterward.
3. Resuspend the bacterial cell pellet in the tube by adding 250 μL of Solution I and vortexing thoroughly.
4. Introduce 250 μL of Solution II to the tube and gently invert it 6-8 times to lyse the bacteria completely.
5. Mix 350 μL of Solution III with the bacterial lysate, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to a clean centrifuge tube.
6. Combine the supernatant from the previous step with 0.35 times the volume of anhydrous ethanol and mix well.
7. Apply the supernatant-ethanol mixture to an adsorbent column, allowing it to stand for 2 min, then centrifuge at 12000 rpm for 1 min, discarding the waste liquid.
8. Wash the column by adding 750 μL of rinse solution, centrifuging at 12000 rpm for 1 min, and discarding the waste solution.
9. Repeat the wash step with 700 μL of rinse solution, followed by centrifugation and discarding the waste solution.
10. Centrifuge the column at 12000 rpm for 2 min and let it sit at room temperature briefly.
11. Place the column in a clean tube, add 40 drops of eluent to the center of the adsorbent membrane, let it stand for 2 min at room temperature, and centrifuge at 12000 rpm for 1 min.
2. Construction of pRED-GFP

A. Construction of pRED-GFP

Goal: Obtain the expression plasmid carrying GFP.

Materials: Designed primers specific to pRED-GFP construction, keeping other materials consistent with the previous procedure.

Procedures: The GFP fragment and the vector were obtained through PCR. Subsequently, a plasmid was constructed using the homologous group method. Followed by transformation into competent E. coli DH5α.

B. Transferring the recombined fragment into DH5α

Goal: Transform the constructed plasmid into E. coli for validation and amplification.

Materials: Consistent with the above.

Procedures: Consistent with the above.

C. Colony PCR Validation

Goal: Verify the transformants.

Materials: Consistent with the above.

Procedures: Consistent with the above.

3. Construction of pRED-Anti-PD-1

A. Construction of pRED-Anti-PD-1

Goal: Construct a functional plasmid containing Anti-PD-1 on the expression plasmid.

Materials: Designed primers specific to pRED-Anti-PD-1 construction, keeping other materials consistent with the previous procedure.

Procedures: Transform the ligated plasmid into E. coli competent cells.

B. Transferring the recombined fragment into DH5α

Goal: Transform the constructed plasmid into E. coli for validation and amplification.

Materials: Consistent with the above.

Procedures: Consistent with the above.

C. Colony PCR Validation

Goal: Verify the transformants.

Materials: Consistent with the above.

Procedures: Consistent with the above.

4. Construction of pRED-Anti-PD-L1

A. Construction of pRED-Anti-PD-L1

Goal: Designed primers specific to pRED-Anti-PD-L1 construction, keeping other materials consistent with the previous procedure.

Materials: Designed primers specific to pRED-Anti-PD-L1 construction, keeping other materials consistent with the previous procedure.

Procedures: Transform the ligated plasmid into E. coli competent cells.

B. Transferring the recombined fragment into DH5α

Goal: Transform the constructed plasmid into E. coli for validation and amplification.

Materials: Consistent with the above.

Procedures: Consistent with the above.

C. Colony PCR Validation

Goal: Verify the transformants.

Materials: Consistent with the above.

Procedures: Consistent with the above.

1. The growth curve test of bacterial strains.

Goal: Assessing the growth status among different bacterial strains.

Materials:

1) ultraviolet spectrophotometer

2) pipette

Procedures:

1. Prepare the growth medium LB according to the specified recipe and sterilize it.
2. Inoculate the medium with the microbial to initiate growth (initial OD₆₀₀ = 0.5).
3. Incubate the culture at the 37 ℃ with continuous monitoring.
4. At regular intervals, remove samples to measure the OD at a specific wavelength using a spectrophotometer.
5. Record the OD values to plot a growth curve showing the growth dynamics over time.
6. Analyze the growth curve to determine the growth phases and growth rate of the microbial culture.
7. Repeat the experiment with appropriate controls and replicates for accurate and reliable results.
8. Interpret the growth curve data to draw conclusions about the growth behavior of the microbial species under the experimental conditions.
2. Fluorescence testing of GFP

Goal: To validate the plasmid's expression, GFP was cloned into the plasmid, followed by testing its expression in the bacterial strain.

Materials:

1) microplate reader

2) pipette

Procedures:

1. Inoculate 12 bacterial strains (4 plasmids with 3 replicates each, starting OD=0.5) and culture at 37°C, incubating under both dark and light-induction conditions.
2. Measure GFP expression intensity at 0, 2, 4, 6, 8, and 24 hours of cultivation (GFP excitation wavelength 488nm; emission wavelength 507nm).
3. SDS-PAGE for Anti-PD-1 and Anti-PD-L1

Goal: Evaluate the protein expression levels of Anti-PD-1 and Anti-PD-L1.

Materials:

1) bacteria culture containing desired protein

2) 50 mL centrifuge tubes

3) beaker containing ice

4) centrifuge

5) sonicator

6) Sangon 12.5% SDS-PAGE Color Preparation kit

7) pipette

8) distilled water

9) electrophoresis buffer

10) protein ladder

11) vertical electrophoresis system

12) Coomassie blue

Procedures:

1) Transfer samples into 50 mL centrifuge tubes and centrifuge at 4000 rpm for 20 mins. Place a centrifuge tube partially submerged in an ice-filled beaker, then place the beaker and tube into the sonicator.

2) Sonicate for 15 mins. Repeat the sonication process for all samples. Centrifuge samples at 4000 rpm for 15 mins. Discard the cell fragments collected at the bottom of the tubes. The remaining supernatant will contain the desired proteins.

3) Combine 2.2 mL of 2X separating gel solution, 2.2 mL of 2X separating gel buffer, and 44 μL of catalyst in a plastic cup using a pipette.

4) Carefully inject the mixture into the casting stand and frame to prevent bubble formation.

5) Add 1 mL of water to level the top surface. Allow the gel to set for 8 mins. Remove the water layer from the top of the gel.

6) Mix 825 μL of 2X stacking gel solution, 825 μL of 2X stacking gel buffer, and 11 μL of catalyst in a plastic cup using a pipette.

7) Fill the cast entirely with the stacking gel mixture, gently insert the comb without introducing air bubbles.

8) Let the gel set for 12 mins, remove the comb carefully, and rinse the wells with electrophoresis buffer.

9) Load the protein ladder into the first well, followed by sample loading in subsequent wells.

10) Transfer the gel into a vertical electrophoresis system.

11) Set the voltage to 120V and run the gel for 90 mins. Stain the gel with Coomassie blue by immersing it for 30 mins.

4. WB for Anti-PD-1 and Anti-PD-L1

Goal: For the detection of target proteins.

Materials:

1) WB kit

2) pipette

Procedures:

1) Cell lysis: Use lysis buffer to break the cells and release proteins.

2) SDS-PAGE gel electrophoresis: Load protein samples onto SDS-PAGE gel to separate proteins based on molecular weight.

3) Transfer proteins from gel to membrane: Use a wet transfer method to transfer separated proteins to a PVDF membrane.

4) Blocking: Block nonspecific binding sites with a protein blocking agent.

5) Primary antibody incubation: Incubate the membrane with a specific primary antibody to allow binding to the target protein.

6) Washing: Wash the membrane with TBST to remove unbound antibody.

7) Secondary antibody incubation: Incubate the membrane with an HRP-conjugated secondary antibody to form a complex with the primary antibody.

8) Washing: Wash the membrane again with TBST to remove unbound secondary antibody.

9) Visualization and imaging: Use a staining method to visualize protein bands, followed by imaging and analysis.

5. ELISA analysis of production of two nanobodies under different lighting times



Goal: Quantitative analysis of the antibody production of two antibody-producing strains under different red light induction conditions.

Materials:

1) microplate reader

2) pipette

3) ELISA KIT

4) red light

Procedures:

1) Inoculate two bacterial strains separately into conical flasks with an initial OD of 0.5, with three replicates for each strain. Subsequently, collect samples at different time points and store the samples in a -20°C freezer.

2) rhTNF-α Coating: Coat microwells with 100 μL per well (2μg/mL, 200ng in total) of PD-1/PD-L1diluted in Coating Buffer (1×PBS). Seal plate and incubate overnight at 4° C.

3) Wash Plate: Aspirate wells and wash 3 times with ≥ 300 μL/well Wash Buffer (1×PBS containing 0.05% Tween20). After last wash, invert plate and blot on absorbent paper to remove any residual buffer.

3) Blocking: Block plates with ≥ 200 μL/well Blocking Buffer. Incubate at RT for 1 hour.

4) Washing Plate: Repeat step (3).

5) Prepare tested sample dilutions in Assay Diluent Buffer.

6) Sample Incubation: Pipette 100 μL of each sample, and control into wells. Seal plate and incubate for 2 hours at RT or 37° C 1hr.

7) Washing Plate: Aspirate/ wash as in step 2, but with 5 total washes.

8) Detection Ab Incubation: Add 100μL of His Tag Mouse Manodonal Antibody (HRP Conjugated)(in Assay Diluent Buffer) to each well. Seal plate and incubate for 1 hour at RT. Note: The Conc. Of anti-His Tag mAb-HRP should be optimized.

9) Washing Plate: Repeat step (8).

10) Detection: Add 100 μL of TMB Substrate Solution to each well. Incubate plate (without plate sealer) for 10-30 minutes at room temperature in the dark. Note: The best color development time will fluctuated based on different exp. context. The longest color development time is not more than 30 minutes.

11) Stop Reaction: Add 100 μL of TMB Stop Solution (650nm) to each well.

12) Data Collection: Read absorbance at 650 nm within 30 minutes of stopping reaction.

6. ELISA analysis of production of two nanobodies under different lighting intensities

Goal: Quantitative analysis of the antibody production of two antibody-producing strains under different lighting intensities conditions.

Materials:

Consistent with the above.

Procedures: Inducing bacterial strains by irradiation with red light at different power levels, keeping other materials consistent with the previous procedure.

7. ELISA analysis of production of two nanobodies different oxygen conditions

Goal: Quantitative analysis of the antibody production of two antibody-producing strains under different oxygen conditions.

Materials:

Consistent with the above.

Procedures: Fermentation in conical flasks under different oxygen conditions (with baffle and without baffle, keeping other materials consistent with the previous procedure.