E N G I N E E R I N G

Design:

Methanol dehydrogenase (ADH) derived from Stearothermophilus was selected, the gene sequence of this enzyme was obtained from NCBI database, and two different label protein fusion modes were designed, namely, the CL2 gene was fused to the N-terminal (2A) and C-terminal (A2) of ADH protein, respectively.

Figure 1, diagram of methanol dehydrogenase construction. A: N-terminal fusion of CL2 gene (A2); B: C-terminally fuses the CL2 gene.

Build:

For the first two weeks, we mainly use a brainstorming approach to discuss each student's ideas and get a comprehensive understanding of the IGEM competition by studying the official IGEM website in detail. Every Sunday night, we hold team meetings, which are designed to get the ideas of the team members to exchange and spark innovation. The discussion process was very relaxed and the team members showed great enthusiasm, so the meeting went very smoothly.

Figure 2, schematic diagram of plasmid construction. A. pET28a-ADH-CL2 A2; B. pET28a-CL2-ADH (2A)

Test:

We transferred two different expression plasmids into receptor cell BL21 respectively. After the target protein was expressed by engineering bacteria, the bacteria was broken up, and the target protein A2 was purified by nickel column, but no 2A was found after purification.

Figure 3,SDS-PAGE test results. A. 2A SDS-PAGE test result; B. A2 SDS-PAGE test result

Then we tested the activity of A2 at different PH states by UV spectrophotometer (hyperlink of detection method), but unfortunately, A2 did not show activity.

FIG. 4 shows the activity test results of A2 at different PH. (A) The activity test results of A2 under PH6.0; (B) A2 activity test result at PH6.76; (C) A2 activity test result at PH7.37; (D) A2 activity test result at PH7.88

Learn:

We suspect that during the inoculation of 2A, we inserted the hybrid bacteria into the culture medium instead of our engineered bacteria, and thus did not obtain the target protein. Therefore, we plan to re-inoculate 2A; After discussion with the instructor, we found that our control group was not completely set up, we only set up the control group without enzyme, but with substrate, and the control group without enzyme and without substrate, so we planned to re-express A2, and then set up a more comprehensive control group to test A2 activity.

Design:

We plan to retranslate 2A to verify that we inoculated a hybrid in cycle 1. In addition, we set up a more comprehensive control group to test the activity of A2.

Build:

After discussion with the instructor, we designed a comprehensive control group (FIG. 1) to test the activity of A2 more rigorously.

Figure 5: Control group components

Test:

We successfully obtained engineering bacteria with expression plasmid, and obtained our target protein 2A through bacterial crushing and protein purification operations, and tested the activity of 2A, but unfortunately, 2A was still not active.

Figure 6:2A transformation and SDS-PAGE test results. (A) 2A conversion results; And (B) 2A SDS-PAGE test results

Figure 7:2A activity test results

In addition, we retested the activity of A2 and found that A2 had some activity at a PH of 5.97. However, what is puzzling is that we tested the light absorption value at 340nm of the control group with only enzyme and no substrate, and found that its value also increased to a certain extent. Theoretically, when there is no substrate reaction with enzyme, there should be no change in light absorption value.

(A) (B)Figure 8.A2 test results

Learn:

We thought about the reason and found that when PH was 5.97, white precipitates were produced in the test cupola. We analyzed that the PH was close to the isoelectric point of protein (A2), which caused the condensation of A2, thus increasing the light absorption value. We checked the isoelectric point of methanol dehydrogenase and found that its PI was about 6.02, which confirmed the correctness of our idea. Therefore, we need to retest its activity away from the isoelectric point A2. In addition, we thought that the addition of label CL2 might affect the enzyme's active center at the 5 'end, so we gave up 2A.

Design:

We retest A2's activity by resetting a series of PH gradients around 0.5-1 away from the A2 isoelectric point. In addition, since methanol itself has an inhibitory effect on the activity of A2, we also tested the activity of A2 at different substrate concentrations.

Build:

We set PH: 5.59 6.52 7.06 7.57; Substrate concentration: 1mM, 3mM, 5mM to test the activity of A2.

Figure 9: Activity test groups of conditions

Test:

The results showed that A2 showed no activity in any of the 12 groups under different conditions. (The figure below shows only the data with a substrate concentration of 3mM)

Figure 10: A2 activity test results at substrate concentration of 3mM. (A) Activity curve at PH5.58; (B) activity curve at PH6.52; (C) activity curve at PH7.06; (D) activity curve at PH7.53

Learn:

We found that at substrate concentration of 3mM and PH of 7.06 and 7.53, the light absorption value has a high initial value. We wonder if the enzyme reaction speed is too fast so that we do not observe an upward trend curve. Therefore, we plan to add different enzyme quantities to the test system to continue to explore the activity of A2.

Design:

We set A2:10ul, 20ul, 40ul, substrate concentration of 3mM,PH 7.06 to test the activity of A2.

Build:

The activity test system of each experimental group is shown in the figure below

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Figure 11: Different group test system

Test:

We re-tested the activity of A2 according to the designed experimental group, and the results were shown in the figure below. We successfully observed a curve with an upward trend.

Figure 12: A2 activity test results for different volumes. (A) Add 10ul volume test results; (B) 20ul volume test results; (C) 40ul volume test result

Learn:

We successfully obtained the highly active methanol dehydrogenase A2, which is one step closer to the success of our entire project

Design:

The team selected formaldehyde dehydrogenase (TcFALDH) derived from thermophilic actinomycetes, obtained the gene sequence of the strain from NCBI, and designed a label protein junction location fusion mode --CL7 protein is attached to the N-terminal of TcFALDH (T7).

Figure 1: Schematic diagram of the construction of formaldehyde dehydrogenase, Tc-FALDH-CL2(label fusion at the N-terminal)

Build:

The gene sequence of formaldehyde dehydrogenase (TcFALDH) derived from thermoactinobacteria was optimized according to the codon preference of Escherichia coli and synthesized by Wuhan Jinkairi Bioengineering Co., LTD. The TcFALDH gene fragment and CL7 gene fragment were obtained by PCR amplification. CL7 was attached to the N-terminal of TcFALDH and cloned into pEt28a vector by DNA assembly technique mediated by T5 exonuclidenase. The recombinant expression plasmid verified by sequencing was named PET28A-TCFALDH-CL7 (T7).

Figure 2: pEt28a-TcFALDH-CL7 plasmid map (T7)

Test:

We transferred the expression plasmid into receptor cell BL21-1 and added 0.5 mM IPTG at 18℃ for 16 h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low-temperature ultra-high pressure continuous flow cell crusher, the supernuant was collected by centrifugation, and the target protein was purified by NI-NTA column. The protein eluent was then detected by SDS-PAGE. Then the protein eluent was ultrafiltration concentrated, and then the activity of T7 was detected by ultraviolet spectrophotometer

Figure 3: T7 gel electrophoresis results (where the number below KDa represents molecular weight, S represents supernatant, P represents precipitation, F represents perforation, and W represents whole cell fluid; The numbers at the top of the image represent different concentrations (mM) of imidazole eluents.)

Figure 4: T7 activity test results

Learn:

Due to operational error, the failure to add the protease inhibitor PMSF during the bacteria-breaking process resulted in reduced or even no activity of the protein, so we had to repeat cycle 1.

Design:

We plan to re-transform T7 to achieve our desired goal

Build:

The original expression plasmid is transformed into the engineered bacteria to make it express the target protein

Test:

We successfully obtained the engineered bacteria with the expression plasmid, and tested whether the engineered bacteria expressed the target protein by SDS-PAGE. Unfortunately, the results of this gelatin run showed that we did not get the target protein.

Figure 5: T7 gel electrophoresis results (where the number below KDa represents molecular weight, S represents supernatant, P represents precipitation, and W represents whole cell fluid; The numbers at the top of the image represent different concentrations (mM) of imidazole eluents.)

Learn:

We found that there were more target proteins in the precipitation, but less target proteins in the supernatant. Through communication with the instructor and experts, we agreed that the expression level of engineered bacteria was too high, so the target proteins did not fold correctly and the inclusion bodies were formed. Therefore, we plan to replace a methanol dehydrogenase to continue the experiment.

Design:

The team re-searched for a formaldehyde dehydrogenase (PsFALDH) derived from unclassified Pseudomonas, obtained the whole genome of the strain from NCBI, and attached CL7 to the N terminal of PsFALDH

Figure 6: Schematic diagram of formalin dehydrogenase construction. FALDH-CL7(label fusion at n-end)

Build:

The gene sequence of formaldehyde dehydrogenase (PsFALDH) from unclassified Pseudomonas was synthesized by Wuhan Jinkairi Bioengineering Co., LTD after optimizing according to the codon preference of Escherichia coli. The PsFALDH gene fragment and CL7 gene fragment were amplified by PCR. CL7 was attached to the N-terminal of FALDH and cloned into pEt28a vector through DNA assembly mediated by T5 exonuclidenase. The recombinant expression plasmid verified by sequencing was named PET28A-FALDH-CL7 (F7).

Figure 7: pET28a-FALDH-CL7 plasmid map

Test:

We transferred the expression plasmid into receptor cell BL21-1 and added 0.5 mM IPTG at 18℃ for 16 h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low-temperature ultra-high pressure continuous flow cell crusher, the supernuant was collected by centrifugation, and the target protein was purified by NI-NTA column. The protein eluent was then detected by SDS-PAGE. Then the protein eluent was ultrafiltration concentrated, and then the activity of F7 was detected by ultraviolet spectrophotometer

Figure 8: (A) F7 conversion results; (B) F7 SDS-PAGE test result

Figure 9: F7 activity test results at different PH. (A)F7 activity test curve at PH6.52; (B) Activity test curve of F7 at PH7.37; (C) Activity test curve of F7 at PH8.05; (D) Activity test curve of F7 at PH8.37

Figure 10: (A)F7 at different temperatures; (B) Activity diagram of F7 at different PH

Learn:

In this cycle, we explore the enzyme activity under different ph gradients, the optimal ph is 8.05, and in our gradient selected the optimal ph, the activity of different temperatures of the enzyme detection, found that the highest at 40℃, 50℃, 60℃ is still active, indicating that our enzyme has a certain heat resistance. However, through communication with the teacher, we found that no control group was set up in the experiment, so we plan to set a comprehensive control group to continue to explore the activity of F7.

Design:

We designed a more comprehensive control group to further explore the activity of F7

Build:

We discussed with the instructor and designed a negative control that only added enzyme and ph solution or pbs buffer to reduce the influence of enzyme itself on the change of absorbance value and measure the activity of F7 more accurately.

Test:

We transferred the expression plasmid into receptor cell BL21-1 and added 0.5 mM IPTG at 18℃ for 16 h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low-temperature ultra-high pressure continuous flow cell crusher, the supernuant was collected by centrifugation, and the target protein was purified by NI-NTA column. The protein eluent was then detected by SDS-PAGE. Then the protein eluent was ultrafiltration concentrated, and then the activity of F7 was detected by ultraviolet spectrophotometer

Figure 11: (A) F7 SDS-PAGE test results; (B) F7 activity test results at different PH values

Learn:

After removing the value of the negative control, we found that the change of abs value decreased a lot, indicating that the enzyme itself would also affect the change of light absorption value, and the enzyme would denaturate under the influence of different ph values, resulting in the change of light absorption value. F7 has a slightly better activity at alkaline PH. There is still some gap between the enzyme activity obtained by us and the expected target, so we plan to change the conditions of the culture process to improve the expression and activity of the enzyme. We sought help from our instructor, who suggested that we use different receptor cells with different induction concentrations.

Design:

We are going to set different conditions to find the optimal conditions for F7

Build:

We're going to set up several different experimental conditions in order to achieve our goal. Here are the conditions:

Figure 12: Components of each experimental group

Test:

In these experiments, we transferred the expression plasmid into the receptor cells, crushed the bacteria after the engineering bacteria expressed the target protein, purified the bacteria by nickel column, ran protein gel to detect whether the target protein was expressed and the corresponding purification gradient. The target protein F7 was obtained from the protein eluent of the target protein purification gradient by ultrafiltration. Then we detected the activity of F7 by ultraviolet spectrophotometer

Figure 13, cell transformation results of different receptive states.

Then we tested the activity of A2 at different PH states by UV spectrophotometer (hyperlink of detection method), but unfortunately, A2 did not show activity.

Figure 14: Gel electrophoresis results of different experimental groups (where the number below KDa represents molecular weight, S represents supernatant, P represents precipitation, F represents perforation, and W represents whole cell fluid; Figures at the top of the picture represent different concentrations (mM) of imidazole eluents.

Learn:

We analyzed the results of these nine experiments and found that the conversion efficiency of BL21 (DE3) -2 receptive cells was significantly higher than that of BL21 (DE3) -1 and E. coli C43 (DE3)PLysS receptive cells. We found that induction with 0.5mM IPTG was significantly better than 0 mM, 0.1 mM, 0.2 mM, and 0.3 mM. Therefore, we finally determined to use BL21 (DE3) -2 receptor cells with an inducer concentration of 0.5 mM IPTG.

Design:

We finally used BL21 (DE3 ) -2 receptive cell transformation, induced by 0.5mMIPTG. In addition, our hydrogenase ABCC has good activity, so we plan to use methylene blue to verify the connection between F7 and ABCC to detect whether there is hydrogen production

Build:

We have designed the experimental scheme of methylene blue detection, as follows:

Figure 15: Methylene blue detection scheme

Test:

The test results are shown below. It can be found that methylene blue in the experimental group changes from blue to colorless, indicating that F7 has good activity.

Figure 15: Results of methylene blue detection

Learn:

The F7 enzyme we obtained is relatively active, reacting with the substrate at room temperature and working with the hydrogenase ABCC to produce hydrogen.

Design:

We selected formate dehydrogenase (FDH) derived from methylomonas xiaoshan, obtained the gene sequence of this enzyme from NCBI database, and designed to fuse CL2 protein to the C-terminal (M8) of ADH protein.

Figure 1, diagram of methanol dehydrogenase construction. A: N-terminal fusion of CL2 gene (A2); B: C-terminally fuses the CL2 gene.

Build:

We selected formate dehydrogenase (FDH) derived from methylomonas koyama and obtained the gene sequence of the enzyme from NCBI, which was synthesized by Wuhan Jinkailui Bioengineering Co., LTD. Then the FDH gene fragment and CL8 gene fragment were obtained by PCR amplification. CL8 was attached to the 3 'end of MDH and cloned into pET28a vector by T5 exonuclidenase mediated DNA assembly technology. The recombinant expression plasmid with accurate sequencing verification was named PET28A-FDH-CL8 (M8).

Figure 2: pET28a-FDH-CL8 plasmid map (M8)

Test:

We transferred pET28a-FDH-CL8 (M8) expression plasmid into receptor cell (BL21-1) and added 0.3mM IPTG at 18℃ for 12h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low temperature ultra-high pressure continuous flow cell crusher, the supernatant was collected by centrifugation, and the target protein was purified by NI-NTA column. However, the protein eluent was detected by SDS-PAGE later, and the concentration of the target protein of M8 was found to be low.

Figure 3: Results of M8 gel electrophoresis

Learn:

We hypothesized that the expression level of the target protein was too low, resulting in less purified protein, and the subsequent preparation was to optimize the induced expression conditions.

Design:

We plan to re-transform M8 and optimize the induced expression conditions

Build:

Test:

We transferred pET28a-FDH-CL8 (M8) expression plasmid into receptor cell (BL21-1) and added 0.5 mM IPTG at 18℃ for 18h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low temperature ultra-high pressure continuous flow cell crusher, the supernatant was collected by centrifugation, and the target protein was purified by NI-NTA column. After that, the protein eluents were detected by SDS-PAGE and M8 protein bands were obtained. After that, ultrafiltration was carried out to concentrate the protein, and the activity of M8 was measured. Although it was active at PH6.43, its activity was low, and the light absorption value only changed by 0.116 in 60s.

Figure 4: M8 gel electrophoresis and activity test results

Learn:

Because the activity of M8 did not meet the expectations, we intend to search for and design a more active formate dehydrogenase

Design:

We selected formate dehydrogenase (FDH) from Saccharomyces cerevisiae, obtained the gene sequence of this enzyme from NCBI database, and designed to fuse CL2 protein to the N-terminal (8F) of ADH protein.

Figure 5: CL8-FDH

Build:

We selected formate dehydrogenase (FDH) from Saccharomyces cerevisiae, and obtained the gene sequence of this enzyme from NCBI, which was synthesized by Wuhan Jinkairui BioEngineering Co., LTD. Then the FDH gene fragment and CL8 gene fragment were obtained by PCR amplification. CL8 was attached to the 5 'end of MDH and cloned into pET28a vector by T5 exonuclease-mediated DNA assembly technology. The recombinant expression plasmid with accurate sequencing verification was named PET28A-Cl8-FDH (8F).

Figure 6: pET28a-CL8-FDH plasmid map (8F)

Test:

We transferred pET28a-FDH-CL8 (M8) expression plasmid into receptor cell (BL21-1) and added 0.5 mM IPTG at 18℃ for 18h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low temperature ultra-high pressure continuous flow cell crusher, the supernatant was collected by centrifugation, and the target protein was purified by NI-NTA column. After that, the protein eluent was detected by SDS-PAGE and M8 protein bands were obtained. After that, ultrafiltration was performed to concentrate the protein, and the activity of M8 was measured. It was found that 8F had the highest activity at PH5.59, and the light absorption value increased by 0.469 within 60s, which was four times the highest activity of M8

Figure 7:8F gel electrophoresis

Figure 8:8F versus M8 activity

Learn:

8F is much more active than M8, so we finally chose 8F and gave up M8. However, the concentration of 8F we obtained is low, and the expression level needs to be improved. Therefore, we will explore the conditions for improving the expression level of 8F in the later stage

Design:

We plan to change the types of receptive states used for expression transformation and the induced concentration to explore the optimal conditions for increasing the expression of 8F.

Test:

By transferring pET28a-FDH-CL8 (M8) expression plasmid into two different receptive cells (BL21-1 and BL21-2), we screened Bl21-2 as the most suitable receptive state by observing the growth of colonies on kanamycin resistant plates. Then, the colonies on the plates with the best growth were selected for expansion culture. Three concentration gradients of 0mM, 0.3mM and 0.5mM were set for induction for the same time, and the optimal induction concentration was determined to be 0.5mM according to the turbidity of the bacterial liquid. After that, the bacteria induced by this concentration were broken up and purified to obtain the target protein 8F, and then its concentration and activity were measured. It was found that the concentration and activity were improved. When 8F was at PH6.76, the light absorption value changed by 1.080 in 60s, which was 2.3 times that before.

Figure 9:8F gel electrophoresis results

Figure 10: Comparison of 8F activity after optimization with 8F activity in cycle 4

Learn:

We have obtained the optimal conditions for the expression of the target protein, and obtained the highly active formate dehydrogenase. At this end of the cycle, we have broken through the difficulties one after another, which is a big step forward from the success of our experiment!

Design:

The NADH-dependent [Fe-Fe] hydrogenase (hyd1ABC) gene sequence from Trophomonas wolfeifera was optimized according to the codon preference of Escherichia coli, and three gene fragments, hydA, hydB and hydC, were obtained by PCR amplification and T5 exonuclidenase mediated DNA assembly technique. CL9 was connected to the N-terminal of hydC subunit to obtain the fragment CL9-HYDC (CC); The HydEFG gene sequence from Clostridium acetonbutanol was optimized according to the codon preference of Escherichia coli, and then PCR amplification was used to obtain the hydE, hydF and hydG gene fragments, respectively. We hoped to co-express HydABCC and HydEFG genes. In order to obtain mature [FeFe] -hydrogenase, and then construct hydrogen production system

Using T5 exonuclease mediated DNA assembly technology, hydA, hydB and hydCC fragments were inserted into the pETTrio vector skeleton to form the Pettrio-CL9-hydabCC expression plasmid. Subsequently, vector ligations mediated by T5 exonuclides were performed, and gene fragments hydE, hydF and hydG were fused with pRSFDuet vector skeleton to construct PRSFDUet-Hydefg chaperone promoting HydABCC expression vector.

Figure 1. Schematic diagram of vector construction: (A): pETTrio-CL9-HydABCC plasmid map,(B): pRSFDuet-HydEFG plasmid map

Figure 2: Results of agarose-gel electrophoresis of hydrogenase and chaperone recombinant plasmid. Note: 1/pETTrio-CL9-HydABCC, 2/ RSFDuet-HydEFG

Test:

Using methylene blue REDOX titration, it is possible to determine whether hydrogen is produced by observing the change of color of the reaction system. At the beginning of the reaction, methylene blue is added for titration and the blue color is disappeared as the standard.

Figure 3: Purification results of hydrogenase detected by SDS-PAGE

Figure 4: Methylene blue verifies hydrogenase ABCC activity

Learn:

This experiment successfully verified that [Fe-Fe] hydrogenase in the hydrogen production system can catalyze the reaction to produce hydrogen using NADH as a substrate.

Design:

The corresponding CE2, CE8, and CE9 truncated mutants CL2, CL8, and CL9 were designed from the E. coli CE7 nuclease inactivated truncated mutant CL7 based on the high similarity of the amino acid sequence of CE2, CE8, and CE9 to CE7. And obtained the corresponding inhibitors Im2, Im7, Im8 and Im9, and constructed the artificial protein scaffold Scaf-CIQ.

Figure 1: Conformation diagram of A.CC7 /IM7; B.ec 7/DNA conformation map; C.CE7 and CL7 protein sequence homologous alignment

Figure 2: Scaf-CIQ

Build:

The Scaf-CIQ gene fragment of the protein scaffold was amplified by PCR reaction, and the Im2, Im7, Im8 and Im9 modules were connected in series by Linker using T5 exonuclease-mediated DNA assembly technology. Scaf-CIQ was sequentially connected from 5 'end to 3' end and cloned into pET28a vector. The recombinant expression plasmid with accurate sequencing verification was named PET28A-SCAF-CIQ (N23).

Figure 3: pET28a-NTScaf-6His plasmid map (N23)

Test:

We transferred pET28a-NTScaf-6His (N23) expression plasmid into receptor cell BL21 and added 0.5 mM IPTG at 18℃ for 16 h. After induction, the bacteria were collected by centrifugation, the cells were crushed by low temperature ultra-high pressure continuous flow cell crushing machine, the supernatant was collected by centrifugation, and the target protein was purified by NI-NTA column. After that, the protein eluent was detected by SDS-PAGE and the target protein of N23 was obtained.

Figure 4: pET28a-NTScaf-6His (N23) gel electrophoresis results

Learn:

We have successfully obtained the protein scaffold N23. We plan to use four enzymes in combination with the protein scaffold to test whether the protein scaffold has a catalytic effect on the reaction system by methylene blue.

Design:

We combined the protein scaffold with methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase and hydrogenase, and tested whether the protein scaffold could improve the efficiency of hydrogen production by methylene blue.

Build:

We plan to fix A2, F7, 8F and N23 by incubating them at room temperature for 10 minutes, and hope to screen out well-combined artificial multi-enzyme complexes through molecular sieve.

Test:

We combined A2, F7, 8F with N23 to obtain a complete artificial multienzyme complex through molecular sieve. We then compared the reaction efficiency of scaffolds with and without scaffolds by using Acalblue. It was found that the Aqua blue color lightened immediately after the addition of the protein scaffold, while it took a while for the non-Aqua blue system. Experiment

Figure 5: Gel electrophoresis results after molecular sieve chromatography

Figure 6: methylene blue test results

Note: Total: NADH production in the Coenzyme NADH regeneration system combined with scaffold protein; Scaf- : NADH production from the Coenzyme NADH regeneration system alone. Figure 7. Effect of SCAF - on enzyme activity of Coenzyme NADH regenerating system

Learn:

By observing the color change, the methylene blue faded faster in the system species added with the protein scaffold, indicating that the protein scaffold could improve the efficiency of hydrogen production, which verified the feasibility of our scheme. In addition, we also compared the efficiency of NADH production with and without protein scaffolds, and found that the rate of NADH production increased by 15.8 times after the addition of protein scaffolds. At this point, our molecular cloning experiment is complete, and we will continue to explore the botanical effects of hydrogen-rich water.

Design:

We explored the effect of protein scaffolds on the activity of individual enzymes

Build:

We mixed the protein scaffold with A2, F7, 8F, HydABCC and other molar ratios, incubated at room temperature for 10min, and then detected the activity of the single enzyme and the enzyme assembled with the protein scaffold respectively.

Test:

The test results are as follows. We found that the protein scaffold can promote the activity of A2, F7 and HydABCC, but inhibit the activity of 8F.
4. The effect of artificial protein scaffold system on the activity of 4 enzymes
(1) The effect of artificial protein scaffold system on A2 activity

Note: a) : Kinetic curves of methanol dehydrogenase (CL2-ADH + Scaf) binding protein scaffold; b) : kinetic curve of methanol dehydrogenase (CL2-ADH). FIG. 8 Kinetic curve of methanol dehydrogenase

(2) Effect of artificial protein scaffold system on F7 activity

Note: a) : The kinetic curve of formaldehyde dehydrogenase (FALDH-CL7 + Scaf) binding protein scaffold; b) : kinetic curve of formaldehyde dehydrogenase (FALDH-CL7). FIG. 9 Kinetic curve of formaldehyde dehydrogenase

(3) The effect of artificial protein scaffold system on 8F activity

Note: a) : Kinetics curve of formate dehydrogenase (CL8-FDH + Scaf) binding protein scaffold; b) : kinetics curve of formate dehydrogenase (CL8-FDH). Note: a): Kinetic curve of formate dehydrogenase binding protein scaffold; b): Kinetic curve of formate dehydrogenase. FIG. 10 Kinetic curve of formate dehydrogenase

(4) Effect of artificial protein scaffold system on HydABCC activity

Note: a) : Kinetics curve of [Fe-Fe] hydrogenase (HydABCC + Scaf) binding protein scaffold; b) : [Fe-Fe] hydrogenase (HydABCC) kinetic curve. FIG. 11 Kinetics curve of [Fe-Fe] hydrogenase

Learn:

Although the protein scaffold has an inhibitory effect on 8F activity, it can still show activity in artificial multi-enzyme complex assembly system.

Design:

We investigated the effect of hydrogen-rich water on seed germination of lettuce and Chinese cabbage. The detailed experimental scheme can be found in our experiment section.

Build:

In order to verify the effect of hydrogen-rich water on plants, we selected Chinese cabbage and lettuce seeds, soaked them for 5h, covered them with moist gauze, placed them in a constant temperature incubator at 28℃, and recorded the germination of the seeds.

Test:

Our experimental results are as follows: hydrogen-rich water has no obvious effect on the germination of lettuce and Chinese cabbage seeds. We wonder whether it is because the seeds themselves and the selected varieties have a high germination rate, so the promotion effect of hydrogen-rich water on the germination of plants is masked.

Figure 12: Effect of hydrogen-rich water on seed germination

Learn:

Unfortunately, we have not observed that hydrogen-rich water can promote the germination of cabbage and lettuce seeds, so we have abandoned this indicator and plan to explore other aspects of plant growth to study the botanical effects of hydrogen-rich water.

Design:

We have designed an experiment on the effect of hydrogen-rich water on plant roots. Please refer to our experiment part for details of the experiment scheme.

Build:

In order to explore whether hydrogen-rich water will affect the root growth of plants, we transplanted the sprouted Chinese cabbage seeds treated with water and hydrogen-rich water respectively. During the plant growth process, we will continue to use hydrogen-rich water and water for treatment, and record the change of their root length at different time periods during the plant growth process.

Test:

The results of our experiment were shown in the figure below. We recorded the root length of the plants for 3 days and 5 days of growth. We found that the overall root system of Pak choi treated with hydrogen-rich water was longer than that treated with water, which was very surprising to us, indicating that hydrogen-rich water does promote plant growth.

Figure 13: Effect of hydrogen-rich water on plant roots (3 days of growth)

Figure 14: Effect of hydrogen-rich water on plant roots (5 days of growth)

Figure 15: Effects of hydrogen-rich water on plant roots (3 days and 5 days of growth)

Learn:

By comparing the results of using hydrogen-rich water and RO water treatment, we found that hydrogen-rich water does promote the root growth of plants.

Design:

We have designed an experiment on the effect of hydrogen-rich water on fresh weight of plants. The detailed experimental scheme can be found in our experiment section.

Build:

In order to further study the botanical effect of hydrogen-rich water, we transplanted the seeds of Chinese cabbage after germination, and designed 4 experimental groups, which were water treatment, water and fertilizer treatment, hydrogen water treatment, and hydrogen water and fertilizer treatment.

Table 16: Treatment of the four groups

Test:

The results of the experiment are shown in the figure below. After 10 days of cabbage growth, the fresh weight of each group of plants was recorded.

Figure 17 Effect of hydrogen-rich water on plant growth (7 days of growth)

Figure 18: Fresh weight data of plants in each group (7 days of growth)

Learn:

We found that 20ml HRW combined with 1.0g fertilizer was more effective than 20ml RO water combined with 1.0g fertilizer, so reducing fertilizer use with hydrogen-rich water may be a viable option.