3.1 The T5 exonuclease system is shown in Table 3.1

     The vector construction system includes: BamH Ι, Nde Ι double enzyme digestion after gel recovery to obtain the vector skeleton, PCR obtained gene fragments (the template has been eliminated with Dpn I), plasmid vector backbone and gene fragments are measured by NanoDrop nucleic acid concentration and reserved. The vector construction process of this experiment is as follows, taking the construction of trisubunit HydABCC hydrogenase expression vector as an example (Figure 3.1).

Table 3.1 T5 digestion reaction system

Component	volume(μL)
Gene fragment of interest	By gene and vector molar ratio 3:1 addition
plasmid backbone	By gene and vector molar ratio 3:1 addition
T5 exonuclease (diluted 10-fold)	0.5
NEB buffer 4	0.5
ddH2O	Up to 5

     1. Add the mixture containing the above reaction system to an ice bath in a centrifuge tube for 5 min and thaw the competent cells at 4 °C.
     2. In the clean stage, add 50-80 μL of E. coli DH5α to the EP tube, incubate on ice for 30 min, then heat shock at 42 °C for 45 s, add 150 μL of NZY liquid, incubate at 37 °C, 220 rpm for 40 min, coat on LB plates (containing appropriate concentrations of Amp and Kana), and incubate at 37 °C inverted for 12-15 h.
     3. After the single bacteria grow in the plate, select about 8 single colonies in the ultra-clean table and inoculate them into LB liquid (containing appropriate concentrations of Amp and Kana), shake the bacteria at 220 rpm at 37 °C to OD600 to 1; perform colony PCR screening with T7 universal positive and negative primers, select 1-2 positive clones and send them to the corresponding company for sequencing, and plasmid extraction of successfully sequenced recombinants is stored at -20 °C for subsequent protein expression.

Figure 3.1 pETTrio-HydABCC expression plasmid construction flow

     1. Regular conversion: E. coli BL21 (DE3) or C43 overexpress competent cells serve as hosts, and the successfully constructed recombinant plasmid vector described above is used for routine transformation of E. coli, followed by heterologous expression of subsequent recombinant proteins.

     2. Seed culture: pick the transformed positive single colony and inoculate it into 10 mL liquid LK (containing 50 g/mL Kana) or liquid LA (containing 100 g/mL Amp), and then place it in a shaker at 37 °C at 220 rpm to reach 0.6~0.8 OD600.

     3. Seed transfer: inoculate the seed solution in TB medium and culture until OD600 reaches 0.8~1.0.

     4. Induction of expression: IPTG (final concentration of 1 mM) was added to induce the expression of recombinant protein, and the bacterial solution was cultured at 18 °C shaker at 220 rpm for 16~18 h.

     5. Collect bacteria: after the end of induction, 6000 rpm, 4°C, 5 min high-speed refrigeration centrifugation to collect bacteria, 25 mL of TBS resuspension to wash the bacteria, 7000 rpm centrifugation for 5 min, bacteria stored at -80 °C for later use.

7.1 Qualitative and quantitative detection of methylene blue

Methylene blue is a universal titration indicator, which can be divided into blue oxidized methylene blue (MB) and colorless reduced methylene blue (leucoMB) types, as shown in Figure 7.1.

Figure 7.1 Molecular structure of methylene blue and argylene trihydrate

Under normal temperature conditions, hydrogen is difficult to undergo redox reaction with blue methylene blue, and the presence of colloidal platinum can promote its combination with equal molecular weight methylene blue, and the reaction of 1 mol hydrogen and 1 mol blue methylene blue can produce 1 mol colorless methylene blue, as shown in Figure 7.1. The reaction formula is: MB + 2H+ + 2e− = leucoMB. Therefore, we selected colloidal platinum-like methylene blue made by Japan Co., Ltd. for subsequent experiments.

Figure 7.2 Under colloidal platinum catalysis, 1 mol of hydrogen reacts with 1 mol of MB to form 1 mol of leucoMB

According to the above formula, the concentration of hydrogen can be determined by methylene blue titration, and the formula for calculating hydrogen concentration according to the methylene blue kit instructions is:
Hydrogen moles = methylene blue titration droplet number × (17/1000) × (0.3/100)/319.85
hydrogen percentage= methylene blue titration droplet× 0.1 ppm.

This method is used to qualitatively and quantitatively detect hydrogen, and the reaction system is shown in Table 7.1.

Table 7.1 Methylene blue titration system (hydrogenase)

Component	volume（mL)
100 mM NADH	0.05
HydABCC	0.1
PBS（PH7.0）	Up to 1

Table 7.2 Methylene blue titration system (artificial multi-enzyme complex)

Component	volume（mL)
Artificial multi-enzyme complex	0.1
100 mM NAD+	0.05
methanol	0.1
PBS(pH 7.0)	Up to 1

If the solution contains other redox substances that will interfere with the accuracy of the titration color, therefore, this method is only suitable for the quantification of hydrogen in redox-free systems, so we use gas chromatography to accurately quantify hydrogen in the system.

Determination of germination rate
(1) Experimental materials: Chinese cabbage seeds, lettuce seeds, germinating box, hydrogen-rich water, gauze
(2) Experimental procedure

① The seeds of Chinese cabbage and lettuce were soaked in hydrogen-rich water and purified water for 5h, respectively, and then placed in germination boxes (20-30 seeds per box). Each treatment was repeated 5 times.

② Cover the seeds with wet gauze and place them in an incubator at 30℃ for germination. If the gauze is dry, spray the wet gauze with hydrogen-rich water and pure water

③ Statistical germination potential after 3 days, statistics of germination rate every day.

(3) Data analysis: Compare the results of hydrogen-rich water and purified water treatment
Second, the effect of hydrogen-rich water on plant growth
(1) Experimental materials: Chinese cabbage seedlings, lettuce seedlings
(2) Experimental steps:

① After germination, 25 seedlings treated with hydrogen-rich water and tap water were selected and transplanted into the soil

② Use hydrogen-rich water to spray twice a day, a total of 10 times, once in the morning and once in the afternoon, and cover the whole plant with each spray

③ The root length of the plant was measured 5 days later.

④ Seedling quality was measured after 5 days.

(3) Data analysis The indexes of plants treated with hydrogen-rich water and tap water were compared