C O N T R I B U T I O N S

Contribution

Improved new parts

Those parts above are improvement for the parts：HrpR Gene (BBa_K1014001), HrpS Gene (BBa_K1014000) and Promoter hrpL (BBa_K1014002) uploaded by iGEM13_HIT-Harbin. The sequences are different from what they uploaded, and it's been quantitatively verified.
In our project，we designed the Amp30E amplification device consists of hrpR (BBa_K4226001), hrpS (BBa_K4226002) , and hrpL promoter (BBa_K4226003) . HrpR protein binds to hrpS protein forming a complex, and the promoter hrpL (BBa_K4226003) is transcriptionally upregulated by hrpR (BBa_K4226001) and hrpS (BBa_K4226002) [1].
We characterized these new parts and added new documentation to it. We hope this will make a contribution to the iGEM community.

New parts

3WJ-Bro

3WJ-Bro is used as a protein- independent reporter system based on a fluorescent RNA aptamer. It is applied as a gene marker in creating a system for reporting the presence and expression of target genes [2].The 3WJ-Bro can be used to ligate fluorescent aptamers to examine the RNA synthesis of target genes at transcriptional level in Escherichia coli cells, obviating the need for accumulation of foreign proteins.

Amp30E Amplification Device

We applied a typical cellular sensor that can be abstracted as a three-stage processor comprising a sensing module that recognizes and transduces external signals into intracellular transcriptional signals, a computing module that modulates the transduced sensor signals, and an output actuating module that executes physiological responses.
We used the promoter of sulA(BBa_K518010), as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR (BBa_K4226001), hrpS (BBa_K4226002) and PhrpL (BBa_K4226003), and the eGFP was used as an output module to test the effect of amplifier module.
The results showed that the Amp30E Amplification Device significantly increased the fluorescence expression of eGFP. This result greatly demonstrates the excellent capability of Amp30E Amplification Device as shown below:

Figure:The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Competent host cell: BL21. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction.

We used the UV induced suicide switch promoter to regulate the expression of the relE toxin protein, but the relE toxin protein has no obvious inhibitory effect on cell growth. Therefore, the Am30E amplification system is assembled upstream of the relE gene, which enhances the inhibition of the relE toxin protein on cell growth.

Figure:The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction.

RNA and protein reporting systems

RNA and protein reporting systems in our project consists of 3WJ-Bro (BBa_K4226000) and mScarlet-I (BBa_K3977002). Firstly, we used a protein- independent reporter system, 3WJ-Bro, based on a fluorescent RNA aptamer, which can be applied as a gene marker in creating a system for reporting the presence and expression of target genes. Besides, mScarlet-I is ideal for use as a fluorescent marker. Our results demonstrated that the reporting systems can report the expression of target genes in Escherichia coli cells, obviating the need for accumulation of foreign proteins. Please see Parts Page for more details.

We have added characterization data to the existing parts：

mSarlet-I	BBa_K3977002	Types	Reporter	http://parts.igem.org/Part:BBa_K3977002

The mScarlet-I gene and the relE toxin gene were fused to construct a fusion gene, in order to detect the amount of relE protein synthesis. We removed the terminator of relE and fused it to the mScarlet-I gene [3].
We designed the genetic circuit that contains sulAp (BBa_K518010), Amp30E (BBa_K4226004), relE gene (BBa_K185047), mScarlet-I (BBa_K3977002) and 3WJ-Bro（BBa_K4226000) , and transferred it into DH10B competent cells. The diagram is shown below:

Gene circuit: pSB1C3-sulAp-Amp30E-relE-mScarlet-I-3WJ-Bro

After bacterial culture and UV induction, we measured the OD600 and fluorescence values under 579/616nm. Then, the fluorescence per OD was calculated.
The curve of experimental group was higher than that of control group (The control group did not contain the Amp30E Amplification Device) as shown in the following figure, indicating the mScarlet-I correctly displayed the increasing tendency of relE protein synthesis under the effect of Amp30E Amplification Device.

Figure: The mScarlet-I gene was fused with relE gene to detect the amount of relE protein synthesis (579/616nm). Control group: pSB1C3-sulAp-relE-mSarlet-l-3WJxBro; experimental group: pSB1C3-sulAp-Amp30E-relE-mSarlet-l-3WJxBro.

This is a time when both science and art are more inclusive. Over the centuries, the degree of symbiosis between humans and technology has become higher and higher. Even as McLuhan said, "the medium extends the human senses." Technology is not outside of human nature but a part of human nature. Art has always been an essential method for human beings to reflect and explore the meaning of life. An artist's mission makes us think about how to re-understand human nature in today's technological age.We also brought the discussion of synthetic biology into the Academy of Arts through discussion and cooperation with teachers.

Our Synbio-art Creative Workshop with Tsinghua and Peking

Our project has successfully attracted the attention of the Chinese art world, for we have shown new viable ways of cooperation between Chinese artists and scientists, especially in the emerging field of synthetic biology, which also shows new possibilities for cooperation between iGEM teams.
We developed a series of artworks based on our iGEM project and turned these works into joint exhibition forms with other iGEM teams: Tsinghua and Peking, to attract public awareness of biological art and synthetic biology. At present, we have established a wide-ranging synthetic biology community in the school. This brought the discussion of synthetic biology into the Academy of Arts through discussion and cooperation with artists and students.

Exhibition effect picture：the Tears of Horus' Eye.

Click here for more exhibition information.

we will promote the widespread attention of synthetic biology methods and perspectives in the global artist community. There is no accepted methodological and theoretical background in the field of bioart. We hope to continue our efforts from iGEM, based on synthetic biology, to set a benchmark for contemporary bioart research, and to make synbio-art a new artistic paradigm for the future of biology!

Other activity photos

Reference

[1] Wan, Xinyi, et al. "Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals." Nature chemical biology 15.5 (2019): 540-548.
[2] Bai, Jiuyuan, et al. "A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering." Nature communications 11.1 (2020): 1-14.
[3] Bindels, Daphne S., et al. "mScarlet: a bright monomeric red fluorescent protein for cellular imaging." Nature methods 14.1 (2017): 53-56.

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