1. RED Homologous Recombination

2. Plasmid Preparation

3. Transformation：Electroporation Procedure,Chemical Transformation

4. PCR

5. SDS-PAGE

6. Preparation of Competent Cells

7. Preparation of Competent Cells for Chemical Transformation

8. Gel Preparation

9. Goldengate

10. Colony PCR

11. Restriction Digestion

12. Microplate Reader Detection

13. Digestion

14. Enzyme Assay

15. Induction

16. Whole-Cell Catalysis

17. Sample Preparation

18. Coffee Grounds Collection and Drying : Coffee Grounds Collection, Coffee Grounds Drying.

19. Caffeine Extraction and Detection from Coffee Grounds: Caffeine Extraction, Sample Preparation, HPLC Detection

20. Attempt to Produce 7-MX Using Coffee Grounds :Extraction of Caffeine as a Production Substrate,Production of 7-MX,Sample Preparation for 7-MX Detection,HPLC Detection

1. Design Homologous Arms and Knockout Fragment: To knockout the target gene, we choose an antibiotic resistance gene (e.g., kanamycin or chloramphenicol resistance gene) as a selection marker. Flank this resistance gene with sequences homologous to the upstream and downstream regions of the target gene (typically about 500 bp on each side). These flanking regions are used as homologous arms.

2. PCR Amplification of Knockout Fragment: Use a plasmid containing the resistance gene (e.g., pKD3 or pKD46) as a template for PCR amplification of the resistance gene fragment with homologous arms.

3. Transformation of Resistance Fragment: Introduce the PCR-amplified resistance gene fragment into bacteria that already carry the λ-RED system via electroporation. Prior to this, induce the expression of the recombination genes in the λ-RED system using arabinose to enhance recombination efficiency.

4. Homologous Recombination: Under the action of the λ-RED system, the exogenous resistance gene fragment will replace the target gene in the bacterial chromosome through homologous recombination, thereby achieving gene knockout.

5. Screening and Verification: Plate the transformed bacteria on selective media containing the appropriate antibiotic to select for resistant colonies. Extract genomic DNA from resistant colonies and verify the gene knockout by PCR or sequencing.

1. Harvest Cells: Transfer the overnight cultured medium to a 1.5 ml EP tube using a pipette. Centrifuge at 10,000×g for 1 min to collect the cell pellet. Discard the supernatant. Repeat this step to enrich the cell pellet.

2. Resuspend Cells: Add 250 μL of Solution I (containing RNase A and stored at 4°C) to the cell pellet. Use a pipette to resuspend the cells until no clumps are visible.

3. Lysis: Add 250 μL of Solution II and gently invert the tube 4-7 times. Incubate for 2-3 min to lyse the cells.

4. Neutralization: Add 350 μL of Solution III and gently invert the tube 4-7 times. Centrifuge at maximum speed (15,000×g) for 10-15 min.

5. Bind DNA: Carefully transfer the supernatant to a blue adsorption column and centrifuge at maximum speed for 1 min. Discard the waste liquid and return the column to an empty collection tube.

6. Wash: Add 500 μL of HBC Buffer (containing isopropanol) to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and return the column to an empty collection tube.

7. Wash Again: Add 700 μL of DNA Wash Buffer to the adsorption column. Centrifuge at maximum speed for 1 min, discard the waste liquid, and repeat this step twice.

8. Dry: Place the adsorption column in an empty collection tube and centrifuge at maximum speed for 2 min to remove any residual wash buffer.

9. Elute DNA: Transfer the adsorption column to a clean 1.5 ml EP tube. Place the tube in a metal bath at approximately 55°C for 3-5 min to dry.

10. Elution: Add 30-50 μL of distilled water to the center of the adsorption membrane. Let it stand at room temperature for 1 min, then centrifuge at maximum speed for 1 min to elute the DNA.

11. Storage: Store the extracted DNA at -20°C.

1. Add the target fragment (greater than 200 ng, preferably not exceeding 5 mL) to 100 mL of competent cells and place on ice for 10-30 min.

2. Sterilize and dry 2 mm electroporation cuvettes under UV in a laminar flow hood for 20 min, pre-chill on ice, and quickly transfer the competent cells to the cuvette, ensuring cells are at the bottom.

3. Perform the following steps quickly: Wipe the outside of the cuvettes dry. For 2 mm cuvettes, use electroporation program Ec2; for 1 mm cuvettes, use program Ec1. After electroporation, immediately add 900-1000 µL of pre-warmed LB at 37°C, gently pipette, and transfer to a 1.5 mL centrifuge tube. Incubate at 30°C on a shaking incubator at 150 rpm for 45-60 min.

4. Plate an appropriate volume onto Kana+Str double antibiotic plates and incubate at 37°C.

1. Thaw competent cells (stored in a -80°C freezer) on ice, add the ligation product/plasmid, and incubate on ice for 20 min.

2. Heat shock at 42°C for 45 sec, then let the cells rest on ice for 2 min.

3. Add 600 µL of LB medium without antibiotics and incubate at 37°C with shaking for 45 min.

4. Centrifuge at 4000 rpm for 2 min, discard an appropriate amount of supernatant, and plate the cells (using both diluted and concentrated samples if needed). Label the plates accordingly.

5. Incubate the plates upside down at 37°C for 12 h.

1. Prepare the reaction mixture in a PCR tube on ice and mix thoroughly using a vortex mixer.

2. Collect any residual liquid on the tube walls using a mini centrifuge.

3. Transfer the PCR tube to the thermal cycler and set the program to initiate the PCR thermal cycling process.

1. Gel preparation:

Assemble the module and secure the gel mold. Prepare the lower gel according to the following recipe, pour it into the mold, and add water to level the surface. Let it sit for 40-60 min until the gel solidifies. Once solidified, discard the water on the surface. Prepare the upper gel according to the recipe, pour it into the mold, insert the comb, and allow it to solidify for approximately 1 h. After solidification, dismantle the mold to complete the gel preparation.

2. Sample preparation:

Collect the induced and uninduced bacterial cultures, measure and obtain 6 OD units of bacterial suspension. Centrifuge at 10,000 g for 10 min, discard the supernatant, and resuspend the pellet in 600 µL of distilled water. Use an ultrasonic cell disruptor to lyse the cells. Centrifuge the lysate at 10,000 g for 10 min to separate the supernatant and pellet. Both fractions are heated at 100°C in a metal bath for 5 min for inactivation, completing the sample preparation.

3. Sample loading:

Prepare the protein buffer solution according to the table above. Place the gel into the electrophoresis apparatus. After checking for leaks with the buffer solution, mix 16 µL of the prepared sample with 4 µL of loading buffer. Load 16 µL of the mixture into the wells of the gel, followed by 4 µL of the protein marker. Then, sample loading is complete.

4. Electrophoresis:

Connect the power supply and run the gel at 80 V for 30 min, followed by 220 V for about 1 h.

5. Staining:

Carefully peel the gel off in water, then place the gel into the staining solution. Boil the solution and then use the heated staining solution to stain the gel at room temperature for 1 h.

6. Destaining:

Place the stained gel in boiling destaining solution and destain it three times.

Composition:

Upper gel:

Lower Gel :

Protein Destaining Solution (500 mL system):

Anhydrous ethanol (75 mL), acetic acid (50 mL), distilled water (375 mL).

5× Protein Buffer Solution (5× Tris-Glycine Buffer; 1 L system):

Tris base (15.1 g), glycine (94 g), SDS (5 g); adjust the pH to 8.3 with precise measurements.

SDS-PAGE Staining Solution (400 mL system):

Coomassie Brilliant Blue R-250 (0.4 g), isopropanol (100 mL), glacial acetic acid (40 mL), distilled water (260 mL); stir thoroughly and filter using filter paper, frequently replacing the paper to remove particulate matter. Prepare two bottles at once, which can be reused.

5×Protein SDS-PAGE Loading Buffer:

(Finally, the volume of distilled water was fixed to 5 mL, and stored at -20 ℃.)

1. Transfer 200 μL of the glycerol stock into 5 mL of LB liquid medium and culture at 30°C, 200 rpm for approximately 12 h. Subsequently, inoculate into LB medium at a 2% concentration and incubate for 0.5 h before adding arabinose to a final concentration of 0.2%.

2. After culturing at 30°C for approximately 2 to 2.5 h, achieving an OD₆₀₀ of about 0.55-0.6 (values significantly higher or lower can severely affect electroporation efficiency), remove the culture and incubate on ice for 30 min.

3. Pre-cool the centrifuge rotor in a freezer for 10 min. Collect the cells by centrifugation using pre-cooled centrifuge tubes and keep on ice. Centrifuge at 4000 rpm for 10 min, aliquoting 50 mL per tube.

4. Using 18 mL of pre-cooled, sterile 10% glycerol per tube, gently pipette while keeping the tube on ice. Aliquot into two 10 mL centrifuge tubes and centrifuge at 4200 rpm for 10 min in a low-temperature centrifuge.

5. Immediately after centrifugation, discard the supernatant. Add pre-cooled, sterile 10% glycerol and then pipette gently while keeping the tube on ice.

6. Repeat steps 4 and 5 three times. For the first two washes, use 5 mL of pre-cooled, sterile 10% glycerol. For the final wash, resuspend in 0.25 mL of 10% glycerol and combine the two tubes into one.

7. Aliquot 100 μL per tube into pre-cooled centrifuge tubes. Proceed with electroporation or store immediately at -80°C. The competent cells can be stored at -80°C for up to six months.

1. From -20°C or -80°C glycerol stock, use a sterile loop or white pipette tip to pick up the bacterial culture. Streak it onto an agar plate and incubate for 12 h (to isolate single colonies).

2. Pick single colonies and inoculate into 5 mL of liquid LB medium using a white pipette tip (or by pipetting up and down). Label the tubes and incubate at 37°C with shaking for 12 h.

3. Transfer 1 mL of the LB culture into 100 mL of LB medium and incubate at 37°C with shaking for about 2 h. Measure the OD until it reaches approximately 0.4 (monitor time carefully).

4. Place the culture into a 16°C shaker and incubate for about 1 huntil the OD reaches 0.5.

5. At this stage, do not use the alcohol lamp. Perform all procedures under strict aseptic conditions on ice. Aliquot 100 mL of the culture into four 50 mL centrifuge tubes and let sit on ice for 30 min to stop active growth.

6. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant, and remove residual liquid using a pre-cooled pipette tip. Add 1 mL of a mixed solution of 80 mM MgCl₂ and 20 mM CaCl₂ to each tube (you may combine centrifuge tubes, ensuring balance).

7. Incubate on ice for 30 min.

8. Centrifuge at 4°C and 4200 rpm for 10 min. After centrifugation, place the tubes on ice, quickly discard the supernatant using aseptic techniques, and remove residual liquid with a pre-cooled pipette tip. Resuspend the pellet in 4 mL of a 15% glycerol and 100 mM CaCl₂ mixture, gently shaking on ice.

9. Aliquot 100 μL into pre-cooled tubes. Record the number of aliquots prepared. Prepare labeling for the storage bags, including preparation date, preparer, competent cell type, and aliquot volume. Place the labeling bags in ice for cooling.

10. Store at -80°C.

1. Prepare agarose gels of appropriate concentration: 1% for detection gels and 2% for recovery gels. Heat in a microwave until the agarose is fully dissolved.

2. Allow the solution to cool to approximately 60°C, then add 1/10,000 Goldview and mix thoroughly.

3. Place an appropriately sized comb into the gel mold. Pour the warm agarose gel into the mold, ensuring the gel thickness is between 3-5 mm. Allow the gel to solidify for approximately 20 min.

4. Once the gel has completely solidified, gently remove the comb and place the gel tray into the electrophoresis chamber. Add 1× TAE buffer so that the buffer level is about 1 mm above the gel surface, ensuring proper conductivity.

Sample Loading:

1. Add the following to the wells: (PCR products + loading buffer) or DNA marker.

Electrophoresis:

1. Cover the electrophoresis chamber and run the gel at 80-120V.

2. When the dye front has migrated an appropriate distance through the gel (approximately 30 min), turn off the power, remove the gel, and observe the DNA bands under a UV lamp.

Gel Imaging:

1. Place the gel onto the gel imaging system and capture an image under ultraviolet light to visualize the gel.

1. Design primers with restriction enzyme recognition and cleavage sites added to both ends of the vector and the target gene fragment.

2. Prepare the reaction system in PCR tubes on ice, and mix thoroughly.Centrifuge to collect the liquid at the bottom of the tube.

3. Centrifuge to collect the liquid at the bottom of the tube.

4. Transfer the PCR tubes to the thermal cycler and set the parameters to initiate the ligation.

1. Prepare the colony PCR reaction system. For a 10 µL reaction volume, use: 1 µL of bacterial suspension, 0.4 µL of upstream and downstream primers, 5 µL of PCR Mix, and 3.2 µL of ddH₂O. If directly picking a colony, add ddH₂O to a final volume of 10 µL. For a 50 µL reaction volume, scale up the components proportionally

2. Set up the PCR program according to the length of the bacterial PCR fragment and run the PCR.

3. Prepare the electrophoresis gel based on the number of bacterial PCR products and the system.

4. Load the bacterial PCR products and an appropriately sized DNA marker into the wells of the gel. Place the gel into the electrophoresis chamber and run the gel at 120-180V.

5. Determine the electrophoresis completion time based on the marker bands. After completion, remove the gel, visualize under UV light, and photograph for analysis.

1. Select appropriate restriction sites based on the plasmid map.

2. Identify the corresponding restriction enzymes and system instructions, and prepare the reaction mixture according to the instructions.

3. Incubate the prepared mixture at the specified temperature in a metal bath or PCR machine for the recommended time.

1. The strain was washed 3 times with water and then washed with M9 medium 2 times to finally obtain the bacterial solution dissolved in M9.

2. Multi-well plate preparation: Sterilize and label the 96-well plate, and add 200 μl of aqueous solutions of different solvents to be tested to the wells.

3. Add 200 μl of bacterial solution dissolved in M9 to each well.

4. Cultivate under suitable conditions.

5. Set the corresponding parameters on the microplate reader to measure the growth of each well strain.

1. Add Dpn I enzyme: Add 1 µL of Dpn I enzyme (10 U/µL) to each 50 µL PCR reaction mixture.

2. Gently mix the reaction mixture to avoid vigorous shaking and incubate at 37°C for 1 h to ensure complete digestion of the methylated template plasmid by Dpn I.

3. After digestion is complete, proceed with bacterial transformation without the need to deactivate DpnI.

1. Wash the bacterial culture three times with water and then wash twice with M9 medium to obtain the final bacterial suspension in M9 medium.

2. Prepare a 96-well plate: sterilize and label the plate, and add 200 µL of different solvents and different concentrations of aqueous solutions to the wells.

3. Add 200 µL of the bacterial suspension in M9 medium to each well.

4. Incubate under appropriate conditions.

5. Set the corresponding parameters on the plate reader and measure the growth of the bacteria in each well.

1. Turn on the UV light in the laminar flow hood and sterilize for 10-20 min.

2. Turn off the UV light, then turn on the fan and lighting. Sterilize and place the required glycerol stocks, antibiotics, and LB medium into the laminar flow hood.

3. Add 5 µL of the antibiotic corresponding to the bacterial strain (the volume of antibiotic added should be 1/1000 of the medium volume) to 5 mL of LB medium in a test tube.

4. Transfer 50 µL of bacterial culture from the glycerol stock (the volume of glycerol stock added should be 1/100 of the medium volume) into the LB medium with the added antibiotic.

5. Turn off the fan and lighting, remove the test tube, and incubate at 37°C with shaking for 12 h.

6. After incubation, prepare ZY medium (shaking flask), 50×M, 50×5052, trace elements, MgSO₄, and antibiotics, and sterilize them before placing them into the sterilized laminar flow hood.

7. Add the following to the ZY medium (shaking flask) using a pipette: 2 mL of 50×M, 2 mL of 50×5052, 200 µL of trace elements, 200 µL of MgSO₄, 100 µL of the corresponding antibiotic, and 1 mL of the bacterial culture. Seal the flask with parafilm and secure it with a rubber band.

8. Remove the flask and incubate at 37°C with shaking for 1-2 h until the OD600 reaches 0.5.

9. After incubation, prepare 20% arabinose and IPTG as needed, sterilize them, and place them into the sterilized laminar flow hood.

10. Add 1 mL of 20% arabinose (final concentration 0.2%) and 100 µL of IPTG to the ZY medium (shaking flask) using a pipette. Seal the flask with parafilm and secure it with a rubber band.

11. Remove the flask and incubate at 25°C with shaking for 18 h.(Fig. 1)

Fig. 1 Flasks for incubation at 25°C

1. Remove the induced shaking flask, take the bacterial culture with the corresponding OD value, and transfer it to a 10 mL centrifuge tube. Centrifuge at 4200 rpm and 4°C for 10 min.

2. Discard the supernatant and add 1 mL of Tris-HCl (pH 9) containing the appropriate substrate concentration of caffeine to the pellet. Mix thoroughly.

3. Transfer the mixed solution to a 100 mL shaking flask.(Fig. 2)

Fig. 2 Whole-cell catalyzed sample vials made

4. Incubate the shaking flask at 20°C with shaking for 18 h.(Fig. 3)

Fig. 3 Thermal flasks for incubation at 25°C

1. Use a pipette to add the appropriate amount of Tris-HCl (pH 9) to the shaking flask to dilute the solution (dilution factor is substrate concentration/2) and mix thoroughly.

2. Transfer 1 mL of the bacterial culture from the shaking flask to an EP tube and centrifuge at 10,000×g for 10 min.

3. After centrifugation, remove the EP tube and use a 1 mL syringe to aspirate the entire supernatant.

4. Attach the syringe to a 0.22 µm filter membrane, and push the syringe to filter the supernatant into a new EP tube. Repeat the process if necessary to ensure complete filtration.

5. Use a pipette to transfer the filtered supernatant into a brown liquid-phase vial. The sample is now prepared and can be stored at -20°C for short-term use.

1. Search for nearby coffee shops and prepare to request coffee grounds.

2. Identify four coffee shops: Ruixing, Starbucks, Kudi, and 818.

3. Organize a team to visit the shops and request coffee grounds from the staff.

1. Spread the coffee grounds from each brand separately in foam boxes.

2. Place the foam boxes in an oven set to 60°C and dry for 48 h.

1. Weigh 5 g of dried coffee grounds and add 100 mL of distilled water. Shake to ensure complete dissolution.

2. Heat the mixture in a microwave until boiling, and allow it to boil for 5 cycles.

3. After cooling, filter the solution using a funnel and filter paper to remove undissolved residues, retaining the filtrate.

4. Transfer 1 mL of the filtrate to a 1.5 mL EP tube using a pipette. Place the EP tube in a centrifuge and set to 15,000 g for 5 min.

1. After centrifugation, remove the 1.5 mL EP tube and use a 1 mL syringe to aspirate all of the supernatant.

2. Attach a yellow filter membrane to the 1 mL syringe and filter the liquid into a new 1.5 mL EP tube. Repeat the process to ensure complete filtration.

3. Use a 1 mL syringe to aspirate the filtered liquid again, preparing for a second filtration.

4. Attach the 1 mL syringe to the yellow filter membrane and perform the second filtration, transferring the liquid into a brown liquid-phase vial. Repeat to ensure complete filtration, then seal the vial.

5. The sample is prepared for HPLC analysis. (If immediate analysis is not possible, store the sample at -20°C.)

1. Use a C8 column with a mobile phase consisting of water, methanol, and acetic acid mixed in a ratio of 85:15:0.5 (v/v).

2. Detect the absorbance peak at a UV wavelength of 580 nm with a column temperature of 35°C.

1. Weigh 2 g of coffee grounds from four different brands (Luckin, Starbucks, 818, Cotti) and place them into four 10 mL centrifuge tubes.

2. Add 5 mL of pH=9 Tris to each centrifuge tube and gently shake until the coffee grounds are dissolved.

3. Open the lids of the four centrifuge tubes. Place every two tubes into a 100 mL Erlenmeyer flask for stabilization.

4. Place the Erlenmeyer flasks in a 100°C water bath and heat continuously to fully dissolve the caffeine from the coffee grounds. After heating for 10 min, remove the flasks and allow them to cool to room temperature.

1. After cooling, close the lids of the centrifuge tubes and centrifuge symmetrically at 4000 rpm, 4°C for 10 min.

2. Transfer the supernatant from the centrifuge tubes into 2 mL EP tubes and centrifuge again at 15,000 g for 10 min.

3. Aspirate 1 mL of the supernatant from the previous centrifugation into a 10 mL centrifuge tube containing 100 OD ndmB Q289A strain. Resuspend uniformly.

4. Take 50 μL of the resuspended culture into a 1.5 mL EP tube as the 0-hcontrol sample. Transfer the remaining culture into a 100 mL Erlenmeyer flask, seal with a breathable film, and ferment for 18 h. Every 2 h, take 50 μL samples for subsequent HPLC sample preparation.

1. Add 50 μL of pH=9 Tris to each of the 10 fermentation samples in 1.5 mL EP tubes for dilution. Mix thoroughly and centrifuge at 15,000 g for 3 min.

2. After centrifugation, use a 1 mL syringe to aspirate all of the supernatant.

3. Attach a yellow filter membrane to the 1 mL syringe and filter the liquid into a brown liquid-phase vial. Repeat to ensure complete filtration, then seal the vial.

4. The sample preparation is complete and ready for HPLC analysis. (If immediate analysis is not possible, store at -20°C.)

1. Use a C8 column with a mobile phase composed of water, methanol, and acetic acid mixed in a ratio of 85:15:0.5 (v/v).

2. Detect the absorbance peak at a UV wavelength of 580 nm with a column temperature of 35°C.