1. Preparation of media

1. LB liquid medium:

1. Weigh out 10 g tryptone, 5 g yeast extract, and 10 g NaCl, then add these components to an Erlenmeyer flask.

2. Dissolve the powder in 1 L distilled water in an Erlenmeyer flask.

3. Mix well and sterilize by autoclaving on liquid cycle at 121 °C for 15 min.

4. Allow medium to cool to room temperature after autoclaving before use.

5. Store prepared LB liquid medium at room temperature or 4 °C until use.

2. LB solid medium

   1. Weigh out 10 g tryptone, 5 g yeast extract, and 10 g NaCl, then add these components to an Erlenmeyer flask.

   2. Weigh out 25 g of LB powder per liter of medium.

   3. Dissolve the powder in distilled water in an Erlenmeyer flask.

   4. Mix well and sterilize by autoclaving on a liquid cycle at 121 °C for 15 min.

   5. Allow medium to cool to 50-60 °C after autoclaving.

   6. Aseptically pour plates in a laminar flow hood. Allow to solidify.

   7. Store prepared LB agar plates upside down at 4 °C until use.

2. Construction of plasmid pETM6-pnar

1. Double enzyme digestion of fragments and vectors

The double digestion system for pnar fragment is shown in the table below:

The double digestion system for vector is shown in the table below:

The enzyme digestion systems are incubated at 37 ℃ for 3 h.

2. Agarose gel electrophoresis

1. Agarose gel preparation

1. Add 1 g agarose powder to 100 mL 1× TAE buffer in a flask.

2. Heat in microwave on high for 2-3 min, swirling occasionally until agarose dissolves completely.

3. Let agarose cool slightly and add 10 μL Ts-GelRed stain. Swirl to mix.

4. Pour agarose into a gel tray with a well comb inserted. Allow to solidify at room temperature for 10 min.

5. Carefully remove the comb and place the gel in the electrophoresis chamber filled with 1× TAE buffer.

1. Electrophoresis

1. Pour about 400 mL TAE into the electrophoresis chamber.

2. Carefully take out the gel and replace it in the electrophoresis chamber.

3. Prepare DNA samples by mixing the 10× DNA loading buffer with the sample at a ratio of 1: 9.

4. Add 5 μL marker in the first well of the gel, then add 5 μL of each sample into the following wells.

5. Turn on the power of the electrophoresis chamber and wait for about 30-40 min.

1. Visualizing DNA bands

1. Take out the gel and place it under the Ultra Slim LED Illuminator

2. Open the Image Lab and scan the gel.

3. Comparison with markers to identify the target DNA bands

3. Fragment extraction

1. Cut the DNA gel:

1. Excise DNA bands of interest quickly using a clean scalpel under UV light.

2. Cut a minimal amount of agarose gel to minimize contaminants.

3. Transfer gel slices into pre-weighed microcentrifuge tubes and determine the weight of gel slices.

1. DNA gel extraction:

1. Add 3 times of sample volume of Buffer B2, and heat at 50 ℃ for 10 min until the gel is completely dissolved.

2. Transfer the solution into columns, centrifuge the tubes at 8,000 ×g for 1 min, and discard the filtrate in the collection tube.

3. Add 500 µL Wash Solution, centrifuge the tubes at 9,000 ×g for 1 min, and discard the filtrate in the collection tube.

4. Repeat the wash step.

5. Place the column in a clean 1.5 mL tube. Add 30 μL Elution buffer to the center of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 ×g for 1 min and keep the DNA solution.

4. Ligation of pETM6-pnar

The ligation system is shown in the table below:

The ligation system is incubated at 16 ℃ for 16 h, followed by 5 min at 65 ℃ to inactivate the ligase.

5. Transformation of recombinant plasmid to E. coli DH5α

1. Add 10 μL of pETM6-pnar plasmid into 100 μL of E. coli DH5α competent cells.

2. Incubate the mixture on ice for 30 min.

3. Heat shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5 min.

4. Add the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

5. Centrifuge it at 9,000 ×g for 1 min, remove 800 μL of supernatant, and resuspend the cells in the remaining LB culture.

6. Spread 50 μL cells onto LB agar plates containing ampicillin and incubate overnight at 37 ℃.

6. PCR identification of transformants

1. Place 50 μL of Lysis Buffer for Microorganism to Direct PCR in a sterilized microtube. Pick a single colony with a sterilized toothpick or lance tip, and place it in the microtube.

2. After heat denaturation at 80 °C for 15 min, centrifuge at low speed and take 1~5 μL of supernatant after lysis as PCR template.

3. The PCR reaction system is shown in the table below:

The PCR reaction condition is shown in the table below:

4. Inoculate the positive transformants in 3 mL LB culture medium containing 50 μg/mL ampicillin at a 37 ℃ shaker with 220 rpm overnight.

7. Plasmid Extraction

1. Column Equilibrium

1. Insert one spin column CP3 into the collection tube.

2. Add 500 μL buffer BL in the tube.

3. Centrifuge the tube at 12,000 rpm for 1 min.

4. Discard the supernatant in the collection tube.

2. Bacteria Extraction

1. Harvest 1-5 mL overnight cultured bacteria into a centrifuge tube.

2. Centrifuge the tube at 12,000 rpm for 1 min.

3. Discard the supernatant.

4. Invert the tube on the absorbent paper to dry.

3. Plasmid Extraction

1. Add 250 μL buffer P1 to the centrifuge tube.

2. Insert the tube into the vortex machine until the bacteria are suspended thoroughly.

3. Add 250 μL buffer P2 to the centrifuge tube.

4. Turn it upside down for 6-8 times temperately.

5. Add 350 μL buffer P3 to the centrifuge tube.

6. Immediately turn it upside down 6-8 times temperately until the solution is uniformly mixed.

7. Centrifuge the tube at 12,000 rpm for 10 min.

8. Aspirate the supernatant and transfer it to spin column CP3 in the collection tube.

9. Centrifuge it at 12,000 rpm for 30-60 sec.

10. Discard the supernatant in the collection tube.

4. Wash

1. Add 600 μL buffer PW to the spin column CP3.

2. Centrifuge it at 12,000 rpm for 30-60 sec.

3. Discord the supernatant in the collection tube.

4. Insert the spin column back into the collection tube.

5. Repeat the wash step.

6. Centrifuge the tube at 12,000 rpm for 2 min.

5. Plasmid Elution

1. Insert the spin column CP3 into a clean microcentrifuge tube.

2. Add 50-100 μL buffer EB to the center of the spin membrane.

3. Place the tube at room temperature for 2 min.

4. Centrifuge it at 12,000 rpm for 2 min.

5. Keep the plasmid solution in the centrifuge tube.

3. Construction of plasmid pETM6-pnar-mCherry

1. Double enzyme digestion of fragments and vectors

The double digestion system for mCherry fragment is shown in the table below:

The double digestion system for vector is shown in the table below:

The enzyme digestion systems are incubated at 37 ℃ for 3 h.

2. Agarose gel electrophoresis

This step is as previously described.

3. Fragment extraction

This step is as previously described.

4. Ligation of pETM6-pnar-mCherry

The ligation system is shown in the table below:

The ligation systems are incubated at 16 ℃ for 16 h, followed by 5 min at 65 ℃ to inactivate the ligase.

5. Transformation of recombinant plasmid to E. coli DH5α

This step is as previously described.

6. PCR identification of transformants

This step is as previously described.

7. Plasmid Extraction

This step is as previously described.

4. Mutation library construction for the pETM6-pnar-RBSx-mCherry plasmid

1. RBS sequences design of the nar promoter

Eight downstream RBS sequences of nar promoter are designed by RBS calculator (https://salislab.net/software/), and the sequences are shown in the following table:

2. PCR Point mutations introduced by whole plasmid PCR

For point mutation by whole plasmid PCR, the following primers are designed:

The reaction system for PCR amplification is shown in the table below:

The reaction procedure for PCR amplification is shown in the table below:

3. Degradation of PCR template with DpnⅠ

The degradation system is shown in the table below:

The degradation system is incubated at 37 ℃ for 30 min, followed by 5 min at 65 ℃ to inactivate the ligase.

4. Blunt-end treatment of PCR product and ligation

1. The PCR products are end-blunted using the DNA Blunting Kit with the following reaction system:

* DNA fragments must be precipitated by ethanol.

2. Keep at 70 ° C for 5 min, then transfer to 37 °C.

3. Add 1 μL T4 DNA Polymerase in the mixture. Mix gently.

4. Reaction at 37℃ for 5 min.

5. Transformation of recombinant plasmids to E. coli BL21(DE3)

(1) Add 5 μL of pETM6-pnar-RBS(1-8)-mCherry plasmids into 50 μL of E. coli BL21(DE3) competent cells, respectively.

(2) Incubate the mixture on ice for 30 min.

(3) Heat shock at 42 °C for 90 sec, and put the mixture to the ice for 4-5 min.

(4) Add the LB culture into the tubes to 1 mL, and shake the tubes at 37 ℃, 220 rpm for 30-60 min.

(5) Centrifuge it at 9,000 ×g for 1 min, remove 900 μL of supernatant, and resuspend the cells in the remaining LB culture.

(6) Spread 50 μL cells onto LB agar plates containing ampicillin and incubate overnight at 37 ℃.

6. PCR identification of transformants

The PCR reaction system and procedure are described previously.

5. Fluorescence intensity detection of RBS mutant library.

1. Inoculate the positive transformants in 3 mL LB culture medium containing 50 μg/mL ampicillin at a 37 ℃ shaker with 220 rpm overnight.

2. Inoculate overnight cultures at a ratio of 1:100 into 25 mL fresh LB culture medium containing 50 μg/mL ampicillin.

3. Incubate at 37 °C, 200 rpm until OD₆₀₀ reaches 0.8.

4. Set the shaker speed to 50 rpm and 30 ℃ to induce protein expression.

5. After induction, mCherry expression is measured every half hour using a fluorescence enzyme marker (excitation light at 580 nm, emission light at 610 nm).

6. All samples are diluted with phosphate buffer solution (PBS) to the appropriate OD₆₀₀ for fluorescence determination.

7. The BL21 (DE3) strain transformed with the mutant promoter plasmid is assayed under the same conditions as the background and subtracted from the total fluorescence to obtain the actual red fluorescence intensity.