Protocol

1

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PCR for target DNA

Thaw Taq, dNTP, primers, template DNA on ice.

To a new PCR tube, add:

Mix the solution thoroughly. Place the tube in a PCR thermocycler and set the program as follows:

• Initial Denaturation: 5 minutes at 95°C
• Loop (35 cycles):
  • Denaturation: 30 seconds at 95°C
  • Annealing: 30 seconds at 60°C
  • Elongation: 1 minute at 72°C
• Final Elongation: 10 minutes at 72°C
• Storage: 10°C

Use 5 μL of the PCR product for electrophoresis and 45 μL for purification.

2

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Qiagen MinElute® Reaction Cleanup

• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature.
• Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 μl pH indicator I to 30 ml Buffer PB or add 600 μl pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of 7.5.
• Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.
• If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.

Procedure

• Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not necessary to remove mineral oil or kerosene. For example, add 250 μl of Buffer PB to 50 μl PCR reaction (not including oil).
• If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
• Place a MinElute column in a provided 2 ml collection tube in a suitable rack.
• To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column.
• Discard flow-through. Place the MinElute column back into the same tube.
• To wash, add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min.
• Discard flow-through and place the MinElute column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed.
• IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
• Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
• To elute DNA, add 10 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.
• If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

3

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Enzyme digestion reaction and reaction of ligation between target gene and plasmid

• Enzyme digestion

  Insert	10ul
  BamHⅠ	1ul
  NdeI	1ul
  10×K buffer	2ul
  ddw	6ul
  total	20ul

  Incubate the tube at 37℃ for about 2hours, then cleanup according to the previous approaches.

• ligation

  Plasmid DNA	1ul
  Target DNA	7ul
  10×reaction buffer	1ul
  T4 DNA Ligase	1ul
  total	10ul

Mixed thoroughly according to above table, and incubate the tube at 16℃ over night. The ligation products were also cleaned according to the previous approaches.

4

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Preparation competent cell of Escherichia coli

After culturing E. coli BL21(DE3) on LB medium, collect the cells by centrifugation. Resuspend the cells in 0.1 M CaCl₂, mix thoroughly, centrifuge again, remove the supernatant, and resuspend the cells in 0.1 M CaCl₂ at 4°C, preparing them for the next step. Typically, cells from 50 mL of LB medium can be resuspended in 2 mL of CaCl₂ solution.

5

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Transform into Escherichia coli

Thaw competent cells rapidly by immersing the frozen tubes in a 37°C water bath after removing them from the -70°C freezer. Draw about 50 μL of the competent cells into a clean tube and add 5 μL of the recombined plasmid. Incubate on ice for about 30 minutes. Then, heat-shock the cells at 42°C for 90 seconds, and immediately return them to ice for 2 minutes. Add 900 μL of LB medium and incubate the mixture in a roller drum at 37°C for 1 hour. Spread 100 μL of the LB medium containing ampicillin on an agar plate, invert the plate when dry, and incubate overnight at 37°C. Validate the transformation by extracting plasmid DNA from the transformed E. coli and performing agarose gel electrophoresis.

Transformation Procedure

LB broth containing the appropriate antibiotic is inoculated with an overnight culture of bacteria, making a 1:100 dilution of the culture. The bacteria are grown at 37°C with shaking (225 rpm) until they reach the early exponential phase (OD600 0.3-0.4). Subsequently, the bacteria are pelleted by centrifugation at 1000 g for 10 minutes at 4°C, and the cell pellet is resuspended in ice-cold TSS to one-tenth of the original volume until the cell suspension is homogeneous. Alternatively, the centrifugation step can be omitted by directly diluting the cells with an equal volume of ice-cold 2× TSS. The TSS-treated cells are incubated on ice for 5-15 minutes and then pipetted into sterile, prechilled polypropylene tubes in 100-μL aliquots.

For transformation, 1 μL (100 pg-1 μg) of plasmid DNA is added per tube, followed by gentle mixing and incubation of the bacteria/DNA mixture on ice for 5-60 minutes. To achieve maximum transformation efficiencies, a 30- to 60-minute incubation on ice is recommended. A negative control, where no DNA is added to the cells, should also be included.

After the ice incubation period, 0.9 mL of LB broth (or TSS) containing an aliquot of the 2.0 M glucose stock solution to yield a final concentration of 20 mM is added to each tube. The tubes are then incubated at 37°C with shaking (225 rpm) for 1 hour. If necessary, the cells are diluted and plated on agar plates prepared with LB broth and the appropriate antibiotic, following standard methods. The plates are incubated at 37°C for 17-20 hours. Transformation efficiency can be calculated by determining the number of transformants per microgram of DNA.

Excess TSS-treated cells can be prepared for storage after the ice incubation period by pipetting aliquots into sterile tubes and immediately freezing the tubes in a dry ice-ethanol bath. The frozen bacteria should be stored at -70°C and used immediately upon thawing on ice.

Development of Procedure

The effect of the chemical components in TSS on inducing DNA uptake by bacterial cells is summarized in Table I. Previously, we examined the effects of varying concentrations of other divalent cations (e.g., Ca²⁺, Mn²⁺, and Zn²⁺) in our assay system. However, concentrations of Mg²⁺ ranging from 20 to 50 mM yielded the highest transformation efficiencies. The effectiveness of PEG with different molecular weights was also evaluated, and the results showed that PEG 3350 and 8000 preparations were equally effective and provided optimal results.

Additionally, we varied the concentrations of PEG, DMSO, and input DNA in transformation mixtures and harvested bacteria at different stages of cell growth to determine the optimal conditions for maximizing the number of transformants. These data demonstrated that under the currently described assay conditions, we consistently achieved transformation efficiencies of 10⁷ to 10⁸ transformants/μg DNA. Moreover, as shown in Table II, several commonly used strains of E. coli were successfully transformable using this method.

6

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Sample preparation for SDS-PAGE

• 5mL of induced products was centrifuged with 10000rpm for 10 min under 4℃, collecting the cells.
• Resuspend the precipitate with 2mL 0.5M Tris-HCl (pH 6.8), then centrifuged with 10000rpm for 5 min, repeat this step twice.
• Resuspend the precipitate with 5mL buffer for ultrasonication under 200W, 1second, with 2 second interval, 30 cycles, total times with 6 min. then centrifuged with 12000rpm for 25 min, collected the supernatant and precipitation respectively.
• Resuspend the precipitate with 80µL ddw, add 20µL 5×loading buffer and 5µL DTT, mixed thoroughly. Prepare another tube with the supernatant with the same procedure. Then boiling in 100℃ for 6min for SDS-PAGE detection.

Procedure of PAGE

Preparation of the separation and concentration gels

AP and TEMED should be the last to added. When it was added, mixed thoroughly immediately, and after the gel polymerization, pull out the comb and wash the sample hole with double distilled water.

• Load 5μL samples, Start electrophoresis under 80V until the bromophenol blue moving into separation gel, then increase the voltage to 100V.
• After electrophoresis, strip the gel and immerge in the fixed solution until the bromophenol blue change to faint yellow; replace the fixed solution with staining solution, then dyeing and decolorizing for 4 hours.
• Removing the staining solution, following another decolonization step, it could be watch and took photo after the band of protein was complete clear. The gel could be stored in distilled water.

7

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Agarose Gel Electrophoresis

• Prepare 100mL 1xTAE buffer with 1g agarose, and boil it three times, shake completely, and waiting for cool.
• Pour the agarose gel into gel tray, assemble gel pouring apparatus by inserting gate into slots.
• Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
• Fill the rig with 1x TAE buffer.
• Load 8μL of DNA maker into lane.
• Mix 1μL of 10x loading buffer with 5μL DNA sample, load them into lane.
• Run at 100V for 30 min.
• Use the Gel imaging system to check the gel and take a picture.
• Deal with the gel carefully as medical waste.

8

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Preparation of E. coli BL21 cell lysate extract

• Choose the single colonies of the engineered bacteria constructed above and inoculate them in 5 ml LB medium, and culture them overnight at 37°C at 220 rpm in a shaker.

• The next day, the medium above was transferred to 300 mL of 2 × YTPG medium, and cultured at 37°C on a shaker at 220 rpm.

• When the OD600 value of the bacterial concentration grows at the later logarithmic period, the bacteria are collected. Centrifuge 10000g for 1 min and remove the supernatant.

• Suspend the bacteria with the precooled S30 buffer, mix well with a shaker, and mix at 4°C and 8000g for 7 min and remove the supernatant. This step is repeated 3 times, and all traces of the supernatant is finally discarded.

• Add 1 mL precooled S30 buffer to every 1g of bacteria and mix well.

• Cells were crushed with a ultrasonic cell breaker, turn on for 2S, turn off for 2S, the total time is 15m, and the temperature alarm is set at 40°C.

• Centrifuge the tube at 4°C, 12000g for 20 min and take the supernatant into a new tube, freeze it in liquid nitrogen, and store it in the refrigerator at -80°C. Ready for use as the lysate extract.

• Note: 2 × YTPG medium:22 mM potassium dihydrogen phosphate, 40 Mm dipotassium hydrogen phosphate, 100 mM glucose, 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride. S30 Buffer: 10 mM Tris-acetate (pH8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, 2 mM DTT.

9

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Preparation of Filter-Paper-Based Sensing Strips

• Put the Whatmann filter paper strips (0.6 × 4 cm) into the culture dish, add 5% bovine serum protein solution to completely cover the paper, and block it overnight at 4°C.

• The next day, discard the 5% bovine serum protein solution in the culture dish, add ddH2O to moisten the filter paper, place the culture dish in decoloration shaker, incubate for 5 min, and discard ddH2O.

• Repeat step 2 and wash the filter paper 5 times.

• Open the lid of the culture dish and place it in the electric constant temperature drying oven. Dry the filter paper for use.

• Spot a volume of 7 μL of E. coli BL21 Δ LacZ cell lysate extract on Whatmann filter paper strips at spots pre-marked with a pencil.

• Put it in an ultra-low temperature refrigerator at - 80°C for 6 h.

• After precooling in the freeze dryer, the paper pieces are sealed with cling film, and the cling film is perforated for ventilation to freeze dry overnight.

• Stored the filter paper sensing strips at 4°C until used.

10

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Build pRSFDuet-1-PdxA-PdxJ-GFP

Reagent

Procedure

Construction of plasmids

• The amplification of the target gene. Sufficient amount of PCR products were amplified by PCR.

• The PCR products and plasmid vectors were digested separately according to the restriction sites designed by the primers.

• Target fragment and vector ligation: the digested PCR product was linked to the plasmid vector by ligase.

• The recombinant plasmids were obtained and ligated into BL21(DE3) competent cells. Positive clones were screened and verified by sequencing.

11

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Protein expression

• The recombinant plasmid was transferred into Escherichia coli Rosetta (DE3) competent cells, and then plated on plates containing 30µg/mL kanamycin and 34µg/mL chloramphenicol.

• Monoclonal cells were selected and cultured in liquid medium containing 30µg/mL kanamycin and 34µg/mL chloramphenicol.

• When the OD value reached 0.6, 0.5mM IPTG was added, and the cells were cultured at 20°C overnight and 37°C for 6h, respectively.

• The cells were collected by centrifugation, the supernatant was discarded, and the cells were collected.

• PBS was added to the collected bacteria suspension, which was fully dissolved using an ultrasonic crusher. It was then centrifuged, and the centrifuged precipitate was dissolved using buffer B. The supernatant and precipitate were processed separately to prepare for SDS-PAGE detection.

12

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Preparation of CFPS

• A single colony of the engineered bacteria constructed above was selected and inoculated in 5 ml of LB medium and incubated overnight at 37°C in a shaker at 220 rpm.

• On the next day, the above medium was transferred to 300 mL of 2 × YTPG medium and incubated at 37°C on a shaker at 220 rpm.

• Bacteria were collected when the 600 OD value of the bacterial concentration increased in the late logarithmic cycle. Centrifuge 10,000 g for 1 min to remove the supernatant.

• Suspend the bacteria with pre-cooled S30 buffer and mix thoroughly with a shaker at 4°C and 8000g for 7 min and remove the supernatant. The procedure was repeated 3 times and finally all traces of supernatant were discarded.

• Add 30 mL of pre-cooled S1 buffer per 1 g of bacteria and mix thoroughly.

• Cells were crushed using an ultrasonic cell disruptor, operating in cycles of 2 seconds on and 2 seconds off for a total duration of 15 min, with the temperature alarm set at 40°C.

• Centrifuge tubes at 4°C for 20 min at 12,000g, remove supernatant into a new tube, freeze in liquid nitrogen and store in a -80°C refrigerator. It can be used as a lysate extract.

• Note: 2 × YTPG medium: 22 mM potassium dihydrogen phosphate, 40 mm dipotassium hydrogen phosphate, 100 mM glucose, 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride. s30 buffer: 10 mM tris acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, 2 mM DTT.

• Prepare 11 mixtures in advance: 9 mM magnesium acetate, 90 mM potassium glutamate, 80 mM ammonium acetate, 57 mM HEPES-KOH, 0.171 mg/mL tRNA, 0.034 mg/mL folate, 2 mM dithiothreitol, 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.

• Cell-free reaction system (1.5mL) was prepared as follows: 600 μL 11 mixtures, 1.2 mM ATP, 0.86 mM GTP, CTP and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvic acid (PEP), 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli BL2121, 0.1 mM PEP, 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli PEP. V/V) E. coli BL21 Δ Lac Z cell lysate extract, 5% (V/V) 20 mg/mL X-gal chromogenic substrate.

• Add 5 nM pRSFDuet-1_PdxA_PdxJ

• Incubate at 37°C for 1 hour and record the colour change of the solution.

Note: Use coloured proteins as indicators, add the appropriate toe-switch plasmid and do not add the X-gal substrate.

13

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Detection of VB6

Key Reagents:

• Extraction Solution: 30 mL × 1 bottle, store at 4°C. This extraction solution contains insoluble matter and should be shaken well before use.
• Reagent One: 5 mL × 1 bottle, store at 4°C.
• Reagent Two: 1.5 mL × 1 bottle, store at 4°C.
• Reagent Three: Powder × 2 bottles, store at 4°C.
• Standard: Powder × 1 bottle, store at 4°C in the dark. Before use, dissolve the powder in 0.822 mL of distilled water to prepare a 5 mg/mL vitamin B6 standard solution. Store the solution at 4°C in a sealed container, avoiding direct sunlight.

Pre-Experiment Preparation:

• Preparation of Mobile Phase A:
  • Dissolve 1 bottle of Reagent Three in 1000 mL of ultra-pure water.
  • Add 0.55 mL of Reagent Two and mix thoroughly to obtain Mobile Phase A.
• Filtration of Mobile Phase A and Methanol:
  • Filter 1000 mL of the prepared Mobile Phase A and methanol (HPLC grade) using filter membranes.
  • Filter the prepared Mobile Phase A using a 0.22 µm water-phase filter membrane.
  • Filter the methanol using a 0.45 µm organic-phase filter membrane.
• Ultrasonication of Mobile Phase:
  • Sonicate the filtered mobile phase for 20 minutes to remove bubbles.
• Preparation of Standard Solutions:
  • Dilute the 5 mg/mL vitamin B6 standard solution using distilled water by serial dilution to prepare standard solutions with concentrations of 16000 ng/mL, 3200 ng/mL, 640 ng/mL, 128 ng/mL, and 25.6 ng/mL of vitamin B6. (Note: The concentrations of the standards are for reference only and can be adjusted based on the actual sample concentration.)
  • Store the standard solutions at 4°C in the dark (sealed). Before testing, filter the solutions using a water-phase syringe filter into brown vials for analysis.

Procedure

Sample preparation：The cells extractive of 10μL samples were removed by centrifugation at 6200×g for 5 min, and the resulting supernatants were used for HPLC analysis of PN production.

• System Setup:
  • Turn on the computer and switch on all modules of the HPLC system.
  • Install the chromatographic column and open the software.
  • In the method group, set the injection volume to 10 µL, column temperature to 30°C, flow rate to 1 mL/min, and fluorescence detector settings to Ex = 293 nm and Em = 395 nm.
  • Set the run time for a single sample to 10 minutes. Save the method group after setting.
• Column Conditioning:
  • Rinse the column with the appropriate mobile phase.
  • Equilibrate the column with Mobile Phase A until the baseline stabilizes, then begin sample injection.
• Standard Solution Analysis:
  • Inject 10 µL of the standard solution. Vitamin B6 should be separated within 10 minutes, with a retention time of approximately 7.7 minutes (note that retention times may vary depending on the system, column, mobile phase pH, and temperature, so this is for reference only).
• Sample Solution Analysis:
  • Inject 10 µL of the sample solution and detect the peak area of vitamin B6 at the corresponding retention time.
• Complete Sample Injection Sequence:
  • (Includes the cleaning and equilibration of the column after each individual sample measurement)

Calculation of Vitamin B6 Content

Plot the standard concentration (ng/mL) on the x-axis and the peak area on the y-axis to create a standard curve for vitamin B6. Substitute the peak area of the sample into the standard curve to calculate the concentration x (ng/mL) of vitamin B6 in the extract.

The specific calculation method is as follows:

Vitamin B6 content (µg/10⁴ cells) = x × V_extraction ÷ cell number (10⁴) × F ÷ 1000 = 0.001 x ÷ cell number × F

Vextraction: total volume of the extraction solution added, 1 mL (0.6 mL extraction solution + 0.1 mL Reagent One + 0.3 mL distilled water). cell number: unit 10⁴, F: dilution factor, The sample tested after dilution needs to be multiplied by the corresponding dilution factor during calculation, 1000: unit conversion factor, 1 µg = 1000 ng.

14

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Determination of Protein Concentration Using NanoDrop Microvolume Spectrophotometer

• Instrument Installation:
  • System Requirements: The computer system should be Windows XP or later; it does not currently support Apple systems. Follow the prompts to install the software.
  • Connect the computer to the instrument using a USB cable. There is no need to repeatedly disconnect and reconnect for long-term use.
• Daily Operation:
  • Open the sampling arm and place the sample to be measured on the lower measurement base.
  • Close the sampling arm and use the computer software to control the instrument. The liquid will automatically form a column between the upper and lower bases.
  • After measurement, open the sampling arm and use lens paper to wipe off the sample liquid from the base surface. (This simple operation effectively prevents cross-contamination, keeping it below one-thousandth.)
• Daily Cleaning:
  • Before and after each measurement, promptly wipe off the sample liquid from the base surface.
  • Rinse the base three times with double-distilled water. You can also clean the base with a 5.25% sodium hypochlorite solution.
• Sample Volume:
  • Nucleic acid water solution: no less than 1 µL.
  • Cell solution: no less than 1 µL.
  • Other solutions: no less than 2 µL.
• Evaporation Effect:
  • During sample measurement, evaporation can slightly affect the results. Generally, the easurement result will increase by 1-2%. This can be clearly observed when measuring the same sample multiple times.
• Sample Recovery:
  • For valuable samples, you can recover the sample using a pipette after measurement for further use.

Procedure

• Turn on the computer, connect the instrument, and open the NanoDrop 3.0 software or double-click the desktop shortcut.
• On the start screen, select “Protein” to enter the protein measurement interface. Add 2 µL of double-distilled water to the sample base, lower the sample arm, and then wipe off the double-distilled water. This step is to clean the sample base of the instrument. (Do not click any program interfaces during washing.)
• Add 2 µL of double-distilled water to the sample base, lower the sample arm, and then click “OK” to initialize the instrument.
• Wipe off the double-distilled water from the sample stage, add 2 µL of the corresponding blank solution (use the same solvent that was used to dissolve the target for the blank solution; if RNA is dissolved in nuclease-free water, use nuclease-free water as the blank solution), and click the “Blank” button in the top-left corner.
• Wipe off the blank solution from the sample base, add the corresponding volume of the protein solution to the sample base, lower the sample arm, enter the sample name in the top-right corner, and then click the “Measure” button in the top-left corner. Wait for 30 seconds, and the measurement results will be displayed on the main interface. Repeat the measurement three times, record the results, and calculate the average value.
• Wipe off the solution from the sample base, add the next sample to be measured, enter the sample number in the appropriate position, and click “Measure.” Repeat this process to sequentially complete the concentration measurements of all samples.
• After all measurements are completed, click the “Show Report” button in the top-left corner to view the results. Click “Save Report” to save the measurement results.