Team:JACOXH China/Poster
Abstract
Soil has always been important to humans, providing a resource that can be used for shelter and food production. Prevention of contaminated soil is closely related to health problems in soil-plant-animal-human system. Yakult is a kind of active lactobacillus beverage, which contains hundreds of millions of active bacteria beneficial to human body. The improved engineering bacteria in this project is just like this kind of beverage, but this "drink" is provided for earthworms to drink. After the earthworms ingest our "Yakult - improved engineering bacteria", it can not only enhance the earthworm's ability to resist heavy metal pollution in the soil, but also help the earthworm to repair the soil and degradation of heavy metals in soil.Earthworms carrying engineered bacteria just like a "walking" biological platform for the detection and remediation of mercury contaminated soil. Multifunctional engineering bacteria were cultured, with MerR-dependent hypersensitive switch, mercury monitor, and mercury stabilizer. Once mercury ions are found in the soil, multifunctional engineering bacteria scattered in the soil can be activated, releasing chromogenic protein and stabilizing mercury ions in the form of HgS with low toxicity. Moreover, considering engineering bacteria move slowly in the soil and is greatly affected by the environment, we choose earthworm as the walking carrier. The engineering bacteria will be reproduced in the earthworm body and move through soil followed with the earthworm to monitor and remedy mercury contaminated soil movably.
Members
Our students comes from 5 high schools in Xuhui District and Pudong New District in Shanghai.Include the following members:iGEM Student Team Member: Shukai Zhang,Tianyu Chen,Bingqing Fu,Zhengtian Guo, Jiayuan Ji,Renfei Shao ,Yuchen Wang,Shiyu Wu,YiFan Xue,Ziyue Yang,Zhihe Zhou.
The instructors of our team come from East China University of Science and Technology and the Youth Activity Center of Xuhui District.Include the following members: iGEM Team Mentor:Yuanchan Luo,Zexuan Li,Kaihe Yang,Haichen Wang,Lijia Shi
The iGEM Team Primary PI comes from Juvenile's Activites Center of Xuhui, Shanghai iGEM Team Primary PI:Yanjun Wu
The instructors of our team come from East China University of Science and Technology and the Youth Activity Center of Xuhui District.Include the following members: iGEM Team Mentor:Yuanchan Luo,Zexuan Li,Kaihe Yang,Haichen Wang,Lijia Shi
The iGEM Team Primary PI comes from Juvenile's Activites Center of Xuhui, Shanghai iGEM Team Primary PI:Yanjun Wu
Project Goals
Construct an effective engineering bacteria, which can effectively detect mercury ions, display color, enrich and finally fix mercury ions. It can effectively degrade organic and inorganic mercury in the soil. The engineered bacteria will be safe, reliable, and low in cost.
Inspiration
Mercury pollution is a typical type of heavy metal pollution in soil, which will cause serious adverse effects on ecosystem and human health. Mercury ion ingestion in the body can cause neurological diseases and damage to the bones, endocrine and immune systems.
In order to make the project get better application, the problem of mercury pollution in cultivated land cannot be solved by simply using engineering bacteria. Because this does not address the limitations of traditional microbial treatments. Then we thought of earthworms, a common ecological engineer in the soil
We used bacillus subtilis (a type of engineering bacteria) to express the thioprotein, hoping to use the engineering bacteria to enrich the excess metallothionein (MT), and then react with the hydrogen sulfide formed by the atLCD protein in earthworms to produce mercury sulfide. Thus, the purpose of fixing mercury in soil can be achieved.
In order to make the project get better application, the problem of mercury pollution in cultivated land cannot be solved by simply using engineering bacteria. Because this does not address the limitations of traditional microbial treatments. Then we thought of earthworms, a common ecological engineer in the soil
We used bacillus subtilis (a type of engineering bacteria) to express the thioprotein, hoping to use the engineering bacteria to enrich the excess metallothionein (MT), and then react with the hydrogen sulfide formed by the atLCD protein in earthworms to produce mercury sulfide. Thus, the purpose of fixing mercury in soil can be achieved.
Methodology
A.MerR-dependent sensor–hypersensitive switch
A sensor is hoped to regulate the module for mercury detection and remediation, which is not only to improve the efficiency of engineering bacteria to remediation mercury contaminated soil, but also to save the resources of engineering bacteria. MerR proteins are chosen to do this!.In addition to improve the detection sensitivity of Hg (II) in mercury contaminated soil,a Hg (II) signal amplification module is supplemented as follows:
B.Mercury ion colorer
In order to show color change to warn farmers or land owners obviously that the land has been contaminated with mercury, we insert a fluorescent protein followed the structural genes of MerR promoter transcription. After the release of the fluorescent protein, it will combine with Hg (II) in soil to express color rendering. Meanwhile, to avoid confusion with the red or yellow of the soil itself and the green of bryophytes on the soil surface, we choose BFP, a blue fluorescent protein.
C.Mercury ion fixation and remediation In the module for mercury detection and remediation, the downstream structural genes of MerR promoter transcription also include the proteins which can fix and remedy Hg (II) in mercury contaminated soil. They will enrich and fix Hg (II) firstly then stabilize Hg (II) to HgS with low biological toxicity. They are mainly composed of three proteins: organomercurial lyase (MerB), metallothionein (MT) and Arabidopsis L-cysteine demercase (AtLCD).
In order to simplify the experiment, step by step, so we designed the whole Hg sensing and fixing system as follows:
C.Mercury ion fixation and remediation In the module for mercury detection and remediation, the downstream structural genes of MerR promoter transcription also include the proteins which can fix and remedy Hg (II) in mercury contaminated soil. They will enrich and fix Hg (II) firstly then stabilize Hg (II) to HgS with low biological toxicity. They are mainly composed of three proteins: organomercurial lyase (MerB), metallothionein (MT) and Arabidopsis L-cysteine demercase (AtLCD).
In order to simplify the experiment, step by step, so we designed the whole Hg sensing and fixing system as follows:
Results and Conclusions
The experiment was delayed due to COVID-19, so there was no experiment. But we have carried out experimental design, and related reagents and primers have been ordered. We will start the experiment when the epidemic slows down.
We are in the process of testing these variables to determine the parameters that will work best for the genetic circuits we have built using BioBricks.
Objectives and Future work
1.This product can be directly put into the mercury contaminated land, and spread through the original or additional earthworms in the soil, and finally achieve the treatment goal of large-scale mercury contaminated land.
2.This product is theoretically very safe, and there is no biosafety risk. However, we will still cooperate with the Chinese Academy of Sciences and other scientific research institutions, conduct relevant tests in their test fields, and then cooperate with related enterprises such as chemical fertilizer and microorganism, so as to realize the industrial production of the product.
3.In addition, our products can still foresee many challenges in reality. For example, how to optimize the treatment efficiency of the product in different soil environment; how to optimize the planting, reproduction and diffusion ability of the product in different kinds of earthworm; how to make the audience of this product understand the product. These are the challenges we will face later.
2.This product is theoretically very safe, and there is no biosafety risk. However, we will still cooperate with the Chinese Academy of Sciences and other scientific research institutions, conduct relevant tests in their test fields, and then cooperate with related enterprises such as chemical fertilizer and microorganism, so as to realize the industrial production of the product.
3.In addition, our products can still foresee many challenges in reality. For example, how to optimize the treatment efficiency of the product in different soil environment; how to optimize the planting, reproduction and diffusion ability of the product in different kinds of earthworm; how to make the audience of this product understand the product. These are the challenges we will face later.
1.Cooperation
On October 5,2020, the JACOXH-CHINA team held a conference with the Shanghai_SFLS_SPBS Group and the OSA group, via video link, to exchange technology and improve the program.
In order to better promote the improvement of our earthworm project, on October 7, 2020, we held a conference with ECUST_ China group from East China University of science and technology .
During the summer vacation, our HP teams visited the Chinese Academy of Sciences and Shanghai Academy of Agricultural Sciences to interview relevant experts and explain our project idea to them. Through two interviews, we have a deeper understanding of the project and planning.
BRIEF DESCRIPTION OF EXPERT:
Zhang Peng, research fellow, Ph. D., center of Excellence in Plant Chinese Academy of Sciences
Zheng Xianqing, associate research fellow, Shanghai Academy of Agricultural Sciences
In addition, we also went to the farm in Chongming, Shanghai, interviewed the farmer, and got an in-depth understanding of the market value of earthworms and project prospects.
In order to better promote the improvement of our earthworm project, on October 7, 2020, we held a conference with ECUST_ China group from East China University of science and technology .
2.Enducation
On October 10th, a science lecture on synthetic biology was held in Nanyang Model Middle School. Our team members introduced the knowledge of synthetic biology and the development of our project.In addition，In order to be able to spread our project, we posted the video of the project on the website（ https://b23.tv/CfNfHy ）/（https://www.bilibili.com/video/BV1Z54y167w4/）。 We hope that through this way, we can let more people pay attention to heavy metal pollution, so that more people can join the road of heavy metal pollution control, and make our earth more and more clean and healthy.3.Questionnaires and interviews
Through investigation, we found consumers' understanding of earthworms is relatively clear and our research is related to it------the earthworm intestinal bacterial species modification project.More than 60% of people know a lot about the effects of heavy metals in the soil on plants and the environment. However, the understanding of earthworms and environmental pollution related issues is less impressive.In the respect of earthworm feed, more attention is being paid to the effects of drugs and their impact on the environment.Most people also expect further improvements in its future biosafety. Our research is secure in terms of safety and cost, and efficiency and safety are our advantages.During the summer vacation, our HP teams visited the Chinese Academy of Sciences and Shanghai Academy of Agricultural Sciences to interview relevant experts and explain our project idea to them. Through two interviews, we have a deeper understanding of the project and planning.
BRIEF DESCRIPTION OF EXPERT:
Zhang Peng, research fellow, Ph. D., center of Excellence in Plant Chinese Academy of Sciences
Zheng Xianqing, associate research fellow, Shanghai Academy of Agricultural Sciences
In addition, we also went to the farm in Chongming, Shanghai, interviewed the farmer, and got an in-depth understanding of the market value of earthworms and project prospects.
Parts
MerR:The mercury-resistant gene operon activates the expression of the mer gene in the presence of Hg2+, and inhibits its expression when Hg2+ is absent.
MerB3 is an organic mercury lyase.It can efficiently cleavage methyl mercury into Hg2+, and can leave most of the lysed Hg2+ in the bacteria.
PmerR:A promoter regulated by the merR protein. The PmerR sequence is the region recognized and bound by the merR protein. In the absence of Hg2+, merR binds tightly to the −35 to −10 region of the promoter (spacer sequence 19-20 bp), making the two elements recognized by RNA polymerase located on both sides of the DNA double helix, making it difficult to recognize and bind, it hinders the transcriptional expression of the mer operon structural gene and itself. In the presence of Hg2+, the merR protein that binds to Hg2+ causes the rearrangement of the promoter, and the binding site of RNA polymerase is exposed, thereby initiating the transcription of mer-related genes.
RBS:Efficient ribosome binding site.Regulating the expression of atLCD.
AtLCD:AtLCD is an L-cysteine desulfhydrylase from Arabidopsis thaliana and a key enzyme for Arabidopsis to produce endogenous H₂S. It can use L-Cys as a substrate and can catalyze the production of H₂S during the reaction.It comes from chromosome 3 of the Arabidopsis genome and is queried in NCBI database.Since this gene comes from the eukaryotic genome, the sequence of AtLCD is optimized for the Bacillus subtilis we used.
MerB3 is an organic mercury lyase.It can efficiently cleavage methyl mercury into Hg2+, and can leave most of the lysed Hg2+ in the bacteria.
PmerR:A promoter regulated by the merR protein. The PmerR sequence is the region recognized and bound by the merR protein. In the absence of Hg2+, merR binds tightly to the −35 to −10 region of the promoter (spacer sequence 19-20 bp), making the two elements recognized by RNA polymerase located on both sides of the DNA double helix, making it difficult to recognize and bind, it hinders the transcriptional expression of the mer operon structural gene and itself. In the presence of Hg2+, the merR protein that binds to Hg2+ causes the rearrangement of the promoter, and the binding site of RNA polymerase is exposed, thereby initiating the transcription of mer-related genes.
RBS:Efficient ribosome binding site.Regulating the expression of atLCD.
AtLCD:AtLCD is an L-cysteine desulfhydrylase from Arabidopsis thaliana and a key enzyme for Arabidopsis to produce endogenous H₂S. It can use L-Cys as a substrate and can catalyze the production of H₂S during the reaction.It comes from chromosome 3 of the Arabidopsis genome and is queried in NCBI database.Since this gene comes from the eukaryotic genome, the sequence of AtLCD is optimized for the Bacillus subtilis we used.
Contribution
The purpose of this project is to construct various expression elements in the beneficial bacteria Bacillus subtilis, so that the mercury sensing protein merR can activate the expression of MerB3 and AtLCD metal proteins by sensing the concentration of Hg in contaminated soil, so as to immobilize organic and inorganic Hg ions. At the same time, earthworm was used as the living bioreactor of B. subtilis to expand the population of B. subtilis, and further improve the immobilization efficiency of the components constructed in B. subtilis to contaminated soil Hg. Based on the original project (Escherichia coli as expression system, MerR as Hg sensing protein, and various amplification elements to detect Hg concentration in soil), this project has made the following improvements:
The original expression system E. coli belongs to human pathogenic bacteria, which is not suitable for release into environmental soil. Now the expression system is replaced by B. subtilis (BS), which is harmless to environment and human and animal, to express each element, so as to lay a foundation for the future application of environmental protection.
Each element in the original project was adapted to be expressed in E. coli system. Now, the expression system is adjusted to BS (Bacillus subtilis). We redesigned the elements suitable for expression in BS, and constructed the diffusion model of Hg in soil.
The original expression system E. coli belongs to human pathogenic bacteria, which is not suitable for release into environmental soil. Now the expression system is replaced by B. subtilis (BS), which is harmless to environment and human and animal, to express each element, so as to lay a foundation for the future application of environmental protection.
Each element in the original project was adapted to be expressed in E. coli system. Now, the expression system is adjusted to BS (Bacillus subtilis). We redesigned the elements suitable for expression in BS, and constructed the diffusion model of Hg in soil.
Model
Acknowledgements and Sponsors
Acknowledgements
Dr. Youyuan Li
Dr. Jianhua Fan
Dr. Hui Wu
Dr. Qiyao Wang
Sponsors
Dr. Youyuan Li
Dr. Jianhua Fan
Dr. Hui Wu
Dr. Qiyao Wang
Sponsors