iGEM 2023 — Team hubu-sky-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# N O T E B O O K

![](https://static.igem.wiki/teams/4927/wiki/banner/notebook.png)

![](https://static.igem.wiki/teams/4927/wiki/notebook/engineering.png)

This is a comprehensive collection of all of the meetings we have ever had, our activities, our successes and our failures. Follow us, 10
undergraduates learning how to navigate the iGEM world, on
our iGEM journey.

Please click on the months to open up into our monthly notebooks!

[APRIL](https://static.igem.wiki/teams/4927/wiki/notebook/4.pdf)

[MAY](https://static.igem.wiki/teams/4927/wiki/notebook/5.pdf)

[JUNE](https://static.igem.wiki/teams/4927/wiki/notebook/6.pdf)

[JULY](https://static.igem.wiki/teams/4927/wiki/notebook/7.pdf)

[AUGUST](https://static.igem.wiki/teams/4927/wiki/notebook/8.pdf)

[SEPTEMBER](https://static.igem.wiki/teams/4927/wiki/notebook/september.pdf)

* Name and purpose
* Design and methods
* Results
* Data analysis
* Conclusion

## Experiment name and purpose

In recent years, a large number of studies have shown that hydrogen-rich water has a positive effect on plant growth. The team hopes to use synthetic biology to produce hydrogen-rich water for crop watering, replacing part of the fertilizer and reducing the amount of fertilizer, thereby protecting the soil and ecological environment.

## Experimental design and methods

The team's experiments are divided into molecular cloning experiment and botanical experiment. Methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, hydrogenase and protein scaffold are obtained through molecular cloning experiment for the production of hydrogen-rich water. And the hydrogen-rich water was used in botany experiment to verify the botanical effect of hydrogen-rich water.

## Experimental results

The team successfully obtained 4 enzymes and successfully produced hydrogen-rich water in the liquid phase system through a cascade reaction of 4 enzymes

## Data analysis

The team used a protein scaffold to combine the four enzymes to form a multi-enzyme complex to improve the hydrogen production efficiency of the entire system. Experimental verification showed that the hydrogen production efficiency of the hydrogen production system using the protein scaffold was 1.58 times higher than that of the free enzyme system and 24.8 times higher than that of the traditional biooptical hydrogen production.

## Conclusion of the experiment

The preparation of hydrogen-rich water catalyzed by whole enzyme method using methanol as substrate provides a new idea for soil protection and fertilizer reduction, which is expected to be put into large-scale agricultural production in the future.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# E N G I N E E R I N G

![](https://static.igem.wiki/teams/4927/wiki/banner/hp1.png)

* ![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/adh.svg)
* ![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/faldh.svg)
* ![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/fdh.svg)
* ![](https://static.igem.wiki/teams/4927/wiki/engineering/qing-hua-mei/hydabc.svg)
* ![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/amc.svg)

## Methanol dehydrogenase

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 1

Design: Methanol dehydrogenase (ADH) derived from Stearothermophilus was selected, the...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 2

Design: We plan to retranslate 2A to verify that we inoculated a hybrid in cycle 1. In addition, we set up a more....

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 3

Design: We retest A2's activity by resetting a series of PH gradients around 0.5-1 away from the A2 isoelectric...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 4

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/success.svg)

## Formaldehyde dehydrogenase

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 1

Design: The team selected formaldehyde dehydrogenase (TcFALDH) derived from thermophilic actinomycetes, obtained...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 2

Design: We plan to re-transform T7 to achieve our desired goal....

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 3

Design: The team re-searched for a formaldehyde dehydrogenase (PsFALDH) derived from unclassified Pseudomonas, obtained...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 4

We designed a more comprehensive control group to further explore the activity of F7 Build: We discussed with the instructor and...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 5

Design: We are going to set different conditions to find the optimal conditions for F7 Bulid: We're going to set...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 6

Design: We finally used BL21 (DE3) -2 receptive cell transformation, induced by 0.5mMIPTG. In addition, our hydrogenase...

![](https://static.igem.wiki/teams/4927/wiki/engineering/success.svg)

## Formate dehydrogenase

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 1

Design: We selected formate dehydrogenase (FDH) derived from methylomonas...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 2

Design: We plan to re-transform M8 and optimize the induced expression conditions Test: We transferred....

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 3

Design: We selected formate dehydrogenase (FDH) from Saccharomyces cerevisiae, obtained the gene sequence...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 4

Design: We plan to change the types of receptive states used for expression transformation and the induced...

![](https://static.igem.wiki/teams/4927/wiki/engineering/success.svg)

## Methanol dehydrogenase

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 1

Design: The NADH-dependent [Fe-Fe] hydrogenase (hyd1ABC) gene sequence from Trophomonas wolfeifera...

![](https://static.igem.wiki/teams/4927/wiki/engineering/success.svg)

## Methanol dehydrogenase

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 1

Design: The corresponding CE2, CE8, and CE9 truncated mutants CL2, CL8, and CL9 were designed from the E. coli...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 2

Design: We combined the protein scaffold with methanol dehydrogenase, formaldehyde dehydrogenase, formate....

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 3

Design: We explored the effect of protein scaffolds on the activity of individual enzymes...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 4

Design: We investigated the effect of hydrogen-rich water on seed germination of lettuce and Chinese cabbage...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle1.svg)

## Cycle 5

Design：We have designed an experiment on the effect of hydrogen-rich water on plant roots. Please refer to our...

![](https://static.igem.wiki/teams/4927/wiki/hp/bottle2.svg)

## Cycle 6

Design：We have designed an experiment on the effect of hydrogen-rich water on fresh weight of plants. The detailed...

![](https://static.igem.wiki/teams/4927/wiki/engineering/success.svg)

## Cycle one

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/1.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/2.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/3.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/4.svg)

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.

Close

## Cycle two

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/5.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/6.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/7.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/8.svg)

(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.

Close

## Cycle three

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/9.svg)

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)
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/10.svg)

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.

Close

## Cycle four

×

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

img
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/11.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-chun-tuo-qing-mei/12.svg)

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

Close

## Cycle one

×

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).

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/1.svg)

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

Build:

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/2.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/3.svg)

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.)

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/4.svg)

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.

Close

## Cycle two

×

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/5.svg)

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.

Close

## Cycle three

×

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/6.svg)

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).

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/7.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/8.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/9.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/10.svg)

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.

Close

## Cycle five

×

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/11.svg)

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.

Close

## Cycle seven

×

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:

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/12.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/13.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/14.svg)

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.

Close

## Cycle eight

×

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:

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/15.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-quan-tuo-qing-mei/15.svg)

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.

Close

## Cycle one

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/1.svg)

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).

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/2.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/3.svg)

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.

Close

## Cycle two

×

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/4.svg)

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

Close

## Cycle three

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/5.svg)

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).

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/6.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/7.svg)

Figure 7:8F gel electrophoresis

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/8.svg)

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

Close

## Cycle four

×

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/9.svg)

Figure 9:8F gel electrophoresis results

![](https://static.igem.wiki/teams/4927/wiki/engineering/jia-suan-tuo-qing-mei/10.svg)

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!

Close

## Cycle one

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/qing-hua-mei/1.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/qing-hua-mei/2.svg)

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/qing-hua-mei/3.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/qing-hua-mei/4.svg)

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.

Close

## Cycle one

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/1.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/2.svg)

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).

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/3.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/4.svg)

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.

Close

## Cycle one

×

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/5.svg)

Figure 5: Gel electrophoresis results after molecular sieve chromatography

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/6.svg)

Figure 6: methylene blue test results

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/7.svg)

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.

Close

## Cycle two

×

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/8.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/9.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/10.svg)

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
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/11.svg)

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.

Close

## Cycle one

×

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/12.svg)

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.

Close

## Cycle two

×

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/13.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/14.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/15.svg)

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.

Close

## Cycle three

×

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.

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/16.svg)

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.
![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/17.svg)

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

![](https://static.igem.wiki/teams/4927/wiki/engineering/dan-bai-zhi-jia/18.svg)

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.

Close

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# E X P E R I M E N T

![](https://static.igem.wiki/teams/4927/wiki/banner/experiment.png)

## Content

# Molecular cloning experiments

## 1. PCR system and method

Table 1.1 PCR system

|  |  |
| --- | --- |
| Component | volume(μL) |
| Gene templates（1ng/μL） | 1 |
| Primer-F(5 nM) | 1 |
| Primer-R(5 nM) | 1 |
| 2×Phanta Max Master Mix | 5 |
| ddH2O | 2 |

Table 1.2 PCR procedures

|  |  |
| --- | --- |
| steps | Time |
| 1：Pre-denaturation98℃ | 5 min |
| 2：denaturation98 C | 20 s |
| 3：anneal 58℃ | 20 s |
| 4：extension 72℃ | according to the clip size（2 kb/min) |
| 5: back to step 2 | 25 cycle |
| 6:Final extension 72℃ | 5min |
| 7:12℃ | 2min |

## 2. The vector digestion system taking pET28a as an example

Table 2.1 BamHⅠ、Nde Ⅰ enzyme digestion system

|  |  |  |
| --- | --- | --- |
| Component |  | volume(μL) |
| pET28a(1ug/μL) |  | 8 |
| CutSmart |  | 5 |
| BamHⅠ |  | 1 |
| Nde Ⅰ |  | 1 |
| ddH2O |  | 35 |
| Total volume |  | 50 |

Table 2.2 BamHⅠ、Nde Ⅰdigestion method

|  |  |
| --- | --- |
| procedure | Time |
| 37℃ | 40 min |
| 80C | 10 min |
| 12°C | 2 min |

## 3. T5 exonuclease mediated vector construction

### 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.

![](https://static.igem.wiki/teams/4927/wiki/experiment/3-1.png)

Figure 3.1 pETTrio-HydABCC expression plasmid construction flow

## 4. Expression of recombinant proteins

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.

## 5. Purification of recombinant proteins

     1. Resuspend the above collected spare bacteria with 30 mL PBS, and at the same time add 1% 100 mM serine protease inhibitor (PMSF) to the bacterial solution, and then use a high-pressure cell disruptor precooled at 4 °C to break the cells 5~8 times (ultrasonic cell disruptor is used for small protein purification) until the bacterial solution is clarified, the working pressure is 1000~1200 W, and after the cell is broken, 14000 rpm, 4 °C refrigeration centrifugation 30 min, obtain the supernatant containing the protein of interest (discard the pellet).

     2. Pipette 3 mL of Ni-NTA into the protein purification gravity column, wash Ni-NTA with 10 mL PBS 3~5 times until alcohol washes, and then use 10 mL of 10 mM imidazole equilibrium to activate Ni-NTA for backup. Move the protein to the purification column, seal the upper and lower openings with the parafilm sealed, and incubate in an 18 °C shaker for 1.5 h.

     3. Place the incubated purification column on the purification rack and use 10 mL of PBS containing 10 mM, 30 mM, 50 mM, 100 mM, 300 mM imidazole to elute protein (G250 detection) until the eluate has no protein residue.

     4. Add 10 μL of 6×SDS-PAGE loading buffer to 50 μL of the eluted protein of each gradient imidazole, heat at 100 °C for 10 min to denature the protein, centrifuge for 1min, and SDS-PAGE detection.

     5. Transfer the protein eluate containing higher purity to the corresponding ultrafiltration tube, wash the ultrafilter tube with NaOH soaking, wash off the NaOH with ultrapure water, and finally equilibrate the ultrafiltration tube with PBS for the concentration of the protein eluent. After the concentration is completed, the concentrate needs to be changed to obtain a relatively pure protein of interest. After ultrafiltration, the target protein was determined by a microplate reader, aliquoted into EP tubes, and liquid nitrogen was snap-frozen and stored at -80 °C.

## 6. Activity determination of recombinant proteins

     The purified protein of interest was subjected to enzyme kinetic activity determination as follows:

### 6.1 Methanol dehydrogenase activity assay

(1) Configure different concentrations of methanol aqueous solution (mM): 0, 1, 2.5, 5, 7.5, 10, 15.
(2) Configure NAD+ at a concentration of 1 mM.
(3) The reaction system is shown in Table 6.11 is shown.

Table 6.1.1 Methanol dehydrogenase activity assay system

|  |  |
| --- | --- |
| Component | volume(μL) |
| Methanol in water | 30 |
| CL2-ADH/CL2-ADH+scaf | 25 |
| 1 mM NAD+ | 30 |
| PBS | Up to 300 |

### 6.2 Formaldehyde dehydrogenase activity assay

(1) Configure different concentrations of formaldehyde aqueous solution (mM): 0, 1, 2.5, 5, 7.5, 10, 15.
(2) Configure NAD+ at a concentration of 1 mM.
(3) The reaction system is shown in Table 6.2.1.

Table 6.2.1 Formaldehyde dehydrogenase activity determination system

|  |  |
| --- | --- |
| Component | volume(μL) |
| Formaldehyde aqueous solution | 30 |
| CL7-FALDH/CL7-FALDH+scaffoldin | 25 |
| 1 mM NAD+ | 30 |
| PBS | Up to 300 |

### 6.3 Formate dehydrogenase activity assay

(1) Configure different concentrations of sodium formate solution (mM) : 0, 100, 200, 300, 400, 500.
(2) Configure NAD+ with a concentration of 1 mM.
(3) The reaction system is shown in Table 6.3.1.

Table 6.3.1 Formate dehydrogenase activity assay system

|  |  |
| --- | --- |
| Component | volume(μL) |
| Sodium formate solution | 30 |
| CL8-FDH/FDH-CL8+scaf | 25 |
| 1Mm NAD+ | 30 |
| PBS | Up to 300 |

### 6.4 Hydrogenase activity assay

(1) Configure NADH with a concentration of 100 mM.
(2) The reaction system is shown in Table 6.4.1.

Table 6.4.1 Hydrogenase activity assay system

|  |  |
| --- | --- |
| Component | volume（mL) |
| 100 mM NADH | 0.05 |
| HydABCC | 0.1 |
| PBS | Up to 1 |

### 6.5 Determination of enzyme activity of coenzyme regeneration system

(1) Configure NAD+ at a concentration of 100 mM.
(2) Configure an aqueous methanol solution with a concentration of 1 M.
(3) The reaction system is shown in Table 6.5.1.

Table 6.5.1 Coenzyme Regeneration System enzyme activity assay

|  |  |
| --- | --- |
| Component | volume(μL) |
| Methanol in water | 30 |
| Three free dehydrogenases/three dehydrogenases+scaf | 25 |
| 100mM NAD+ | 30 |
| PBS | Up to 300 |

(4) The above reaction mixture was added to the cuvette, OD340 was measured by U-3900 to obtain the NADH concentration of the product, and the effect of protein scaffold on the enzyme activity of the coenzyme regeneration system was comparatively analyzed.

### 6.6 Activity assay of artificial multi-enzyme complexes

(1) Configure NAD+ at a concentration of 100 mM
(2) Configure an aqueous methanol solution with a concentration of 1 M
(3) The reaction system is shown in Table 2.15

Table 6.6.1 Artificial multi-enzyme complex activity assay

Component | volume（mL) || Methanol in water | 0.6 |
| Artificial multi-enzyme complex / 4 free enzymes | 0.6 |
| 100 mM NAD+ | 0.3 |
| PBS | Up to 6 |

(4) The above reaction mixing system determined the concentration of product hydrogen by methylene blue redox titration and gas chromatography

### 6.7 Assembly of artificial multi-enzyme complexes

It is known that the artificial protein scaffold system (Scaf-CIQ) and four CL-tag fusion proteins can be bound together by interaction between CL and Im reaction pairs. As shown in Figure 6.7.1, an artificial multi-enzyme complex AMC based on the four-enzyme cascade reaction pathway can be constructed by mixing the two in equal molar proportions and incubating at room temperature for 5 minutes.

![](https://static.igem.wiki/teams/4927/wiki/experiment/note.png)

Note: Part1: CL-tag fusion protein; Part2: Scaffold protein with Im protein as stock price
Figure 6.7.1 Schematic of artificial multi-enzyme complex assembly

## 7. Hydrogen detection method

### 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.

![](https://static.igem.wiki/teams/4927/wiki/experiment/7-1.png)

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.

![](https://static.igem.wiki/teams/4927/wiki/experiment/7-2.png)

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.

## 8. Verification of botanical effects of hydrogen-rich water

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

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# R E S U L T

![](https://static.igem.wiki/teams/4927/wiki/result/result.png)

## Summary

     Our project "Hydrosoilguard" aims to use synthetic biology techniques to produce hydrogen-rich water to replace part of the fertilizer, thereby reducing the use of fertilizer and protecting the soil environment. We have documented in detail the preparation of hydrogen-rich water by four-enzyme cascade catalysis using methanol as a substrate, which you can find on our engineering page. In addition, we have designed 59 different parts, you are welcome to browse our parts page. On our Education and Human Activities page, you can learn how we expand the impact of our projects through a range of educational and practical activities. Click on each "drop" to get more information.

[![](https://static.igem.wiki/teams/4927/wiki/result/1.svg)](https://2023.igem.wiki/hubu-sky-china/education)

Education

1246

participator

[![](https://static.igem.wiki/teams/4927/wiki/result/2.svg)](http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2023&group=HUBU-SKY-China)

Parts

59

innovation

[![](https://static.igem.wiki/teams/4927/wiki/result/3.svg)](https://2023.igem.wiki/hubu-sky-china/human-practices)

HP

200,000

pageviews

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Specific

Outlook

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