We add new documentation to several existing Parts, including DavB (BBa_K3776001), DavA (BBa_K3776002) and YahK (BBa_K4395013). The detailed information is listed as following:

BioBricks	Short description	Contributions from our team
BBa_K3776001	DavB	Characterization of the length of the davB gene, the molecular weight of DavB protein and the activity of DavB to consume L-lysine.
BBa_K3776002	DavA	Characterization of the length of the davA gene, the molecular weight of DavA protein and the activity of DavA to produce 5-aminovalerate.
BBa_K4395013	YahK	Characterization of the length of the YahK gene, the molecular weight of YahK protein and the activity of YahK.

L-lysine monooxygenase (DavB) is an enzyme in the 5-aminivalerate pathway. Here, we characterized the length of the davB gene, the molecular weight of DavB protein and the activity of DavB to consume lysine for future iGEM teams.

The gene of davB was incorporated into plasmid pRSFDuet to obtain the plasmid pRSFDuet-davB. By sequencing, the correct plasmid pRSFDuet-davB was then transformed to E. coli BL21(DE3) (Figure 1).

Figure 1.The transformation of palsmid pRSFDuet-davB into E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-davB was successful obtained (Figure 2).

Figure 2. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-davB.

M represents the band of DNA marker; Lane 1, 2 and 3 represent the band of different colonies containing plasmid pRSFDuet-davB

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davB was inoculated and cultured. We transformed the plasmid pRSFDuet without davB gene into E. coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).

Figure 3. SDS-PAGE analysis of DavB expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet;

lanes 3 and 4：supernatant and precipitation of E. coli BL21(DE3) containing PRSFDuet-davB

DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. We tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).

Figure 4 The enzymatic activity of the whole-cells containing DavB

We tested the activity of DavB by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).

Figure 5. The enzymatic activity of crude enzyme of DavB

5-Aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. Here we characterized the length of the davA gene, the molecular weight of DavA protein and the activity of DavA catalytic production of 5-aminovalerate for future iGEM teams.

The gene of davA was incorporated into plasmid pRSFDuet to obtain the plasmid pRSFDuet-davA. By sequencing, the correct plasmid pRSFDuet-davA was then transformed to E. coli BL21(DE3) (Figure 6).

Figure 6. The transformation of palsmid pRSFDuet-davA into E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-davA was successful obtained (Figure 7).

Figure 7. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-davA

M represents the band of DNA marker; Lane 1, 2 and 3 represent the band of different colonies containing plasmid pRSFDuet-davA

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We transformed the plasmid pRSFDuet without davA gene into E. coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 8).

Figure 8. SDS-PAGE analysis of DavA expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet; lanes 3 and 4：supernatant

and precipitation of E. coli BL21(DE3) containing PRSFDuet-davA

DavA catalyzes 5-aminovaleramide into 5-aminovalerate. We tested its activity by detecting production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 9).

Figure 9 The enzymatic activity of whole-cells congtaining DavA

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-davA was inoculated and cultured. We tested its activity by detecting production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 10).

Figure 10.The enzymatic activity of crude enzyme of DavA

Yahk: YahK, an aldehyde reductase from Escherichia coli, catalyzes the reduction of various aldehydes to corresponding alcohols. Here, we charactered the length of the YahK gene, the molecular weight of YahK protein and the activity of YahK for future iGEM teams.

The gene of YahK was integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-YahK. By sequencing, the correct plasmid pRSFDuet-YahK was then transformed to E. coli BL21(DE3) (Figure 11).

Figure 11.The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)

The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 1500bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-YahK was successful obtained (Figure 12).

Figure 12. Colony PCR results of recombinant strain that containing the plasmid pRSFDuet-YahK.

M represents the band of DNA marker; Lane 1, 2 and 3 represent the band of different colonies containing plasmid pRSFDuet-YahK

The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-YahK was inoculated and cultured. We transformed the plasmid pRSFDuet without YahK gene into E. coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of YahK protein was correct, which was consistent with the expected molecular weight of 38.0 kDa (Figure 13).

Figure 13. SDS-PAGE analysis of YahK expression in E. coli BL21(DE3)

Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containing pRSFDuet; lanes 3 and 4：supernatant

and precipitation of E. coli BL21(DE3) containing PRSFDuet-YahK

YahK is an NADPH dependent aldehyde reductase. The enzyme of Yahk was purified by with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column. Subsequently, we tested its activity to catalyze 1,5-glutaraldehyde by detecting NADPH consumption with the purified YahK enzyme. The results showed that YahK can consume NADPH, indicating that YahK has active enzyme activity towards the reduction of 1,5-glutaraldehyde.

Figure 14.The enzymatic activity of purified YahK

We first discussed and formulated the core values of our project through team brainstorming. Through the feedback provided by society, we used synthetic biology to create cell factories, which focus on bio-manufacturing, aiming to produce bio-based material monomers 1,5- pentanediol(1,5-PDO). Through a series of Human Practices activities, we introduced synthetic biology, iGEM and our project to the public, increasing public acceptance of bio-manufacturing and bio-based materials, and strengthening the concept and awareness of green and low carbon.

We have carried out a series of activities to provide the public with an opportunity to learn about bio-based materials, iGEM and synthetic biology. These activities are primarily showcased and promoted through communication meetings, community events, academic conferences, and other channels. We collaborated with nearby kindergartens, primary schools, high school, and university to conduct science outreach activities. We disseminated knowledge related to synthetic biology and also engaged with nearby communities. Meanwhile, we held a synthetic biology carnival at our team's university. Additionally, we visited university classrooms and organized online exchange activities through Tencent Meeting.

After engaging in exchanges and interviews with experts and scholars, we received professional guidance and actively participated in various social practice activities. This allowed us to bridge the gap between academia and society and receive valuable advice. These diverse perspectives made the project more comprehensive and mature, and also provided us with a strong technical groundwork for our subsequent work. Through exchanges, more researchers have an in-depth understanding of our project, technologies and concepts.

Our team actively utilized the Internet platform to create popular science videos on the Bilibili Video Platform and manages a popular science WeChat public account. Based on our project, team members created popular science videos showcasing biological experiments and original videos with non-woven as the main body, which is mainly about synthetic biology. At the same time, we have also launched WeChat public accounts to promote and popularize knowledge about biology and environmental protection activities. This allows us to reach a wider audience and educate more people about synthetic biology and our research project.

We connected three parts in order to form a cohesive whole. Firstly, we engaged with the thoughts and challenges presented to us by society. Next, we delved into how the involvement of relevant individuals could enrich our projects. Finally, we concluded by highlighting our own contributions to the progress of society. The entire Human Practice section was cohesive, closely connected to society, and had a significant contribution to the world. The multi-channel feedback proves that our project is good and responsible for the world.

Figure 15. “3 cycles” loop of unique design