Team:CSU CHINA/Poster

Poster: CSU_CHINA

Clean the contamination of Cadmium

Presented by Team CSU_CHINA 2020

Hao Zhou, Chen Ziwen, Mingyu Liang, Yanzhe Zhang, Haonan Zhou, Haoran Hu, Jialong Ruan,Tianyuan Xie, Zhang Gengxin, Dongni Hong, Junxi Liao, Wang Xianghe, Yao Xiao, Xiangyu Chen, Yaxian You, Yuyang Peng, Runyu Lyu, Jiayi Pan, Siqi chen, Jiayu Chen, Zhaoxi Wu, Shiyu Lin, Li Tao, Linhu Xiao, Kelvin Zheng Li, Wen Wen, Lina Ma, Enyou Si, Xiangyu Li, Yufan Dong, Bin Zhang, Danyang Wang, Xutong Guo, Jufang Huang, Shanni Li, Jiada Li, Jie Li, Chiyu Li

School of Life Sciences, Central South University, Changsha, Hunan, China

Abstract

Recently, the cadmium-contaminated rice circulating in the market. Long-term intake of cadmium will jeopardize human bodies. To deal with the problem, we utilize engineered Synechocystis as a competent cadmium absorber. Moreover, with blue-ray/antitoxin suicide system, the reformed alga will be appropriately contained. The cadmium can be recycled as the microorganisms will be calcined after absorption. The application of engineered alga will minimize human potential cadmium intake.

Goal

1. To reduce the cadmium content in the water by using the modified Synechocystis.

2. To increase cadmium tolerance of Synechocystis in a high cadmium concentration environment.

3. To avoid environmental pollution caused by the escape of Synechocystis.

Inspiration

Cadmium is a toxic heavy metal element for the human body. Water and soil cadmium pollution is still severe in many areas in China. Cadmium can cause chronic poisoning by accumulating in the human body through the food chain. Data shows that the annual production of “Cadmium Rice” results in million tons of grain discarded and billions of economic losses[1]. However, there is currently no practical way for farmers to absorb the cadmium for agricultural irrigation water. Thus, our project aims to find out a safe and efficient way to solve water cadmium pollution.

Problem: Current status of cadmium pollution

1) Agricultural production

The impact on crops makes reducing cadmium content in water urgently needed to be solved. According to the Bulletin of the National Survey of Soil Pollution, China’s overall soil environment is not optimistic. Soil pollution is severe in some areas, and cultivated land’s soil environmental quality is also worrying. The exceeding rate of cadmium pollutants reached 7.0%, showing a gradual increase from northwest to southeast and from northeast to southwest. It is one of the primary contaminants in cultivated land, woodland, grassland and unused land. There are 20 million \( h{m}^{2} \) of land contaminated by heavy metals in China, accounting for 1/5 of China’s arable land. Among them, the cadmium pollution of farmland is as high as \( 1.3 x {10}^{2} h/{m}^{2} \), involving 25 provinces and cities in China. The annual production of “cadmium rice” is as many as hundreds of millions of kilograms.

Through the interviews with SINOGRAIN Company and farmers, in China, the main way of soil cadmium pollution is the cadmium polluted-water irrigation, the heavy metal polluted rice result in 12 million tons grain discarded and more than 20 billion economic loss, and there is currently no effective way for farmers to absorb the cadmium for agricultural irrigation water they use.

Figure 2 The vast farmland

2) The Source of Pollution

The pollution of cadmium in water bodies mainly comes from surface runoff and industrial wastewater. The cadmium content in the wastewater discharged from the production of sulfuric acid from pyrite ore, and phosphate fertilizer production from phosphate rock is relatively high. Cadmium particles from atmospheric lead-zinc ore and non-ferrous metal smelting, combustion and incineration of plastic products all of these may enter the water. Cadmium is discharged from catalysts, pigments, plastic stabilizers, synthetic rubber vulcanizers, and fungicides that use cadmium as raw materials can also cause water pollution. As for urban water, the cadmium content in drinking water is often increased due to the corruption of containers and pipelines. The discharge of industrial wastewater makes the cadmium content in offshore seawater and plankton higher than that in the distant sea. The cadmium content in the surface water of industrial areas is higher than that in non-industrial areas.

Figure 3 Industrial waste water being discharged

Cyanobacteria accumulates Cadmium

We use Synechocystis, a kind of Cyanobacteria, as the chassis organism to carry out genetic engineering modification and fix it in the calcium-alginate microbeads(Ca-Alg MBs) to immobilized bacteria.

We used the “Transport-Chelate-Redox” System to increase cadmium uptake and cadmium tolerance of Synechocystis, and designed a “Light-Regulated Toxin-Antitoxin” Suicide System to ensure the death of spillover engineering Cyanobacteria and avoid biological pollution.

Besides, using calcium alginate embedding immobilized method to Synechocystis in Ca-Alg MBs can not only immobilize our biological chassis, but also improve the cadmium elimination.

Module 1: "Transport—Chelate-Redox" System

See sessions below for details

Human Practices

Mainstream cadmium removal methods in sewage treatment plants inspired us to use chelating proteins and oxidoreductase in our first module.

(JIN XIA Sewage Treatment Plant)

Transportation-Chelate-Redox

Cadmium contamination of water worldwide is already having a severe environmental impact, especially on crops, such as cadmium rice. In addition, cadmium contamination is likely to cause itai-itai disease and renal tubular injury, after prolonged and/or high exposure may eventually to renal failure, thus, the problem of cadmium pollution of water needs to be solved urgently. We adopt an environmentally friendly biological approach——use Synechocystis sp. PCC6803 with a clear genetic background and a certain degree of tolerance to cadmium as a chassis organism for transformation.

First of all, we translate cadmium, which is enriched on the cell wall through transporter proteins. However, this may cause photosynthesis failure, protein inactivation or oxidative stress in Synechocystis and lead to cell death, making it difficult to achieve high tolerance rate. Therefore, after transporting large amounts of cadmium ions into the cells, we accumulate heavy metal ions and prevent them from being transported out of the cells by chelating proteins. At the same time, we increase the expression of oxidoreductase to remove reactive oxygen clusters in the cells, thus increasing the growth rate of algae under cadmium ion stress. Besides, for high expression of functional proteins, We optimized the codon preferences of the algae, and analyzed the protein expression results.(Figure 1)

Figure1: The core “transport-chelation-redox reaction” system of our project

Parts

According to the needs of the project, we have designed 9 proteins, of which SmtA draws lessons from the design of Team Groningen 2009. Three of them are transporters, three are metal-binding proteins, and three are redox proteins.

Contribution: SmtA

We used SmtA protein designed by Team Groningen 2009,we use it to reduce the free Cd(II) in the cell, to prevent it from activating the downstream oxidative stress system, to prolong the life cycle of cells. (fig1. a)

We expressed SmtA in E. coli and found that the growth condition of E. coli expressed by SmtA was better than that of the control group, which means SmtA could improve the tolerance of E.coli to cadmium(fig1. b).

Figure a：Western blotting results indicating the successful expression in E.Coli

Figure b：Growth curve after successful protein expression

Engineering: hMT-1A, APX2, MntH

We successfully expressed 8 proteins, in which the metal-binding protein hMT-1A, oxoredoxin APX2, transporter MntH worked perfectly as expected. The expression of hMT-1A, APX can improve the cadmium tolerance of Escherichia coli (fig2. a, b), while MntH can transport cadmium on the cell membrane of E. coli. (fig2. c, d)

(b) Growth curves of metal-binding protein hMT-1A expressed E. coli culturing in 10mg/L Cd(II), (c) Growth curves of APX2-expressed E. coli culturing in 10mg/L Cd(II), (d) Cadmium uptake curve of (MntH / MntH & TMCd1 & APX2)-expressed E. coli, (e) Growth curves of (MntH / / MntH & TMCd1 & APX2)-expressed E. coli.

Results

Experiments in Synechocystis

Growth curve of PCC6803

We measure cyanobacteria’s tolerance and cadmium removal ability of different densities while in 0.1 and 2 milligram per liter of cadmium concentrations. We found that the low cadmium group, the cyanobacteria all grew well. Besides, the lower the concentration was, the more algae would be inhibited.

Cd removal rate of PCC6803

We can see that algae absorbed a certain amount of Cadmium, and when algae dead, it will release the previously removed cadmium.

Embed PCC6803 with Ca-Alg MBs

we considered using Calcium-alginate microbeads to immobilize and protect algae. And we can see algae grows well in the calcium-alginate microbeads!

Actually, by the Growth curve of algae within or without MBs in different Cd concentrations, we learned that MBs can protect algae in high concentration, which means it can improve tolerance.

We also observed an interesting thing——immobilizing algae with Calcium-alginate microbead can increase the Cd removal rate distinctly, which means that MBs can not only immobilize and protect the biological chassis but also foster the cadmium elimination in high Cd by acting as a ‘buffer zone’.

Experiments in E.coli

Proteins successfully express in E.coli

Verify the effect of transport、chelate and redox protein

Because of the Corona Virus, we only expressed these parts in E. Coli successfully. We found that when we expressed transport protein MNTH only, it was found to inhibit the growth of E. coli significantly. And when we expressed chelate protein hMT1-A only, it increased the cadmium tolerance and promoted growth. Moreover, when we expressed transporter chelate redox protein at once, it significantly increased the survival rate，and higher than other groups. From the Cadmium removal rate curve, it also shows that expressing these three proteins at once could mostly improve absorption efficiency. So it actually shows that our design for this project is effective and feasible.

Model

We extended module level from cell into the real world through this model. Based on We extended module level from cell into the real world through this model. Based on element analysis, we rebuilt the microbead and cells virtually, and predict its reaction, then get the module optimized.

Model 1: \( {Cd}^{2+} \) flow between environment and microbead

Model 2: \( {Cd}^{2+} \) flow between algae and microbead

Model3: Predict absorption saturation time & Optimization

Model4: Predict rupture time & Optimization

raw cell concentration ${10}^8 cell/{cm}^3$	0.6	0.8	1.0	1.2	1.4	1.6	1.8	2.0
rupture time $/h$	90	63	49	38	31	26	23	22

Module 2: "Light-Regulated Toxin-Antitoxin" Suicide System

See sessions below for details

Human Practices

We found that the toxin-antitoxin suicide system we designed needs to be started with chemical reagents by visiting farmers. These chemical reagents directly put into the farm will affect the growth of rice. Additionally, escaping our engineered Synechocystis sp.PCC6803 into the natural world may also lead to different adverse effects. We gave careful consideration to the farmer’s concern. Based on these Considerations, we designed our blue-light-controlled “toxin-antitoxin” suicide system.

"Toxin-antitoxin" light-controlled suicide system

The escape of engineered synechocystis is one of the major hazards of genetically modified organism, which may lead to different adverse effects such as water bloom and reduction of water polymorphism. Considering these risks, we designed a blue-light-controlled "toxin-antitoxin" suicide system. (Figure 2) In this system we use the RelE/RelB-toxin/antitoxin system and regulate the expression of the toxin RelE through the blue light-regulated promoter. In the presence of blue light, the regularly expressed HTH-LOV-REP blue-regulated protein will homodimerize and specifically recognize the (c120)5 sequence upstream of the blue-regulated promoter via HTH, while inhibiting the expression of toxin RelE via the REP domain. When synechocystis escaped from the blue light environment we have set up, the uninhibited blue light regulatory promoter will initiate the expression of RelE and under the effect of our amplification system, the content of RelE will increase rapidly, as a result the escaped algae will be killed. Meanwhile in order to prevent the small amount of RelE that is still expressed in the presence of blue light from harming the algae, we regularly express the antitoxin RelB to neutralize its toxin to ensure that the toxin RelE does not harm the unescaped Synechocystis sp.PCC6803.

Figure 2:Third-generation system the Blue light regulated suicide system

Model

The model proves workability of the module with specific parameters, avoiding much wet exp work in the rough pandemic times. Our light-controlled suicide system is proven through this model to maintain the sustainability of regular cells and erase detached cells as expected.

System Off	System On

Future

We designed a fly-catcher-like device, a blue light source is placed at the bottom of the center to deactivate safety system in cells. Algae in microbead create oxygen continuously and gas fuse into a bubble or two inside that provide buoyancy, the microbeads could float as the bubbles grow.

Meanwhile, the absorption process is still in progress. One of the main goals of our model is to optimize parameters to match the saturation time and float up time to maximize its efficiency. Once the absorption process is done, we expect the microbeads float on the surface. Once a microbead floats on the surface, it can just float for a while and rupture, then fall into groove on the top of the device waiting for salvage. Hence, the process of absorption and salvage is separated on time and space scales.

Due to COVID-19, the real implementation of the device just stayed at our to-do list. If possible, future CSU China team could continue to develop the project in 2021. We plan to install microelectronics in the device, including LTE and GPS modules and photoresistors, so that the device can sense the current light intensity and access future light intensity information from the local meteorological department by LTE. With the data sent to an app installed on the user's phone, algorithm based on our model inside the app will provide the user with the best salvage time.

Education

Synthetic Biology Day (Changsha No. 21 Middle School)

We held off-line Synthetic Biology Day activity in Changsha NO. 21 middle school, including brainstorming and biosafety contest.

Synthetic Biology Club Courses (YALI High School)

We assisted the students who were preparing for International Biology Olympiads to build up the Synthetic Biology Club. Moreover, we made synthetic biology educational handbooks for them and taught them synthetic biology courses regularly.

Small Molecules Online Courses

We made a series of promotional articles and videos

to introduce synthetic biology and iGEM. We put promotional materials on mainstream social platforms. These original courses cover more than 800 students from 7 secondary schools dispersing in 5 cities.

Promotional Articles and Videos

The promotional article on Synthetic Biology and iGEM that we posted on WeChat has gained more than 10,200 views, 31 remarks, 159 reposts, and 351 likes.

We posted the promotion video on the Weibo platform and received more than 6,700 views, which greatly expanded our project’s visibility.

Reference

[1] Saffar, B., Mehri Ghahfarrokhi, A., Mahnam, K., & Mobini-Dehkordi, M. (2015). Improvement of Cd2+uptake abipty of SmtA protein by Lys/Cys mutation; experimental and theoretical studies. Journal of Biomolecular Structure and Dynamics, 33(11), 2347–2359. doi:10.1080/07391102.2015.1054431

[2] Suleman A, Shakoori AR. Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia cop. J Cell Biochem. 2012;113(5):1616–1622. doi:10.1002/jcb.24030

[3] Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.

[4] Ochoa-Fernandez, R., et al., Optogenetic control of gene expression in plants in the presence of ambient white pght. Nat Methods, 2020. 17(7): p. 717-725.

[5] Saffar, B., Mehri Ghahfarrokhi, A., Mahnam, K., & Mobini-Dehkordi, M. (2015). Improvement of Cd2+uptake abipty of SmtA protein by Lys/Cys mutation; experimental and theoretical studies. Journal of Biomolecular Structure and Dynamics, 33(11), 2347–2359. doi:10.1080/07391102.2015.1054431

[6] Suleman A, Shakoori AR. Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia cop. J Cell Biochem. 2012;113(5):1616–1622. doi:10.1002/jcb.24030

[7] Harms, A., et al., Toxins, Targets, and Triggers: An Overview of Toxin-Antitoxin Biology. Molecular Cell, 2018. 70(5): p. 768-784.

[8] Ochoa-Fernandez, R., et al., Optogenetic control of gene expression in plants in the presence of ambient white pght. Nat Methods, 2020. 17(7): p. 717-725.