Team:NAU-CHINA/Poster

Title and Abstract

Title

Soil lead immobilization magician（SLIM）：earthworms carrying engineered Bacillus subtilis.

Presented by Team: NAU-CHINA 2020

Team members: Guolong Shi, Zhen Cao, Chengzhu Fang, Yingying Han, Yunong Li, Yuting Sun, Shangping Chen, Peng Chen, Jiaxing Sun, Yifei Zhao, Fengzhi Yuan, Yu Cao, Xiuli Wang, Ziye Lv, Ruijie Zhang, Tianrui Song, Yucheng Chen, Ran Zhang, Xinyue Jiang, Lijun Fan, Jiamao Yang.

College of life sciences, Nanjing Agricultural University, Nanjing, China.

Abstract

Lead pollution in soil has brought great losses to agriculture and human health. However, traditional approaches such as physicochemical repair and plant enrichment will cause other hazards and have lower efficiency, respectively. Therefore, it is urgent to develop new strategies for lead pollution treatment. This year, to make up for the deficiency of traditional methods, NAU-CHINA combined earthworms (Eisenia foetida) and bacteria (Bacillus subtilis) to immobilize soil lead. Earthworms was used as carrier of bacteria, and we transformed bacteria to secrete phytase specifically by using oxygen-regulated switch to hydrolyze phytate in the intestines of earthworms. Lead ions can be precipitated into insolubles by combining with phosphate and chloride ions, thus purifying soil. Meanwhile, we added suicide module considering the biosafety of using genetically modified organisms in the environment. Affected by COVID-19, we cannot do experiments. So we collected data from literature and constructed mathematical models to predict results and feasibility.

Problems

As a global-concerned problem, soil lead contamination in farmlands is urgent to resolve, especially in the moderately lead-polluted cultivated lands, which still have some production efficiency. However, traditional solutions all have many limitations.

Physicochemical immobilization combined with phytoremediation

• Disturbance to soil physicochemical property
• Productivity reduction

Microbial remediation

• Short-time treatment
• Low survival rate

Bioremediation

• Lack further eradication
• Accumulated toxicity is harmful to earthworms

This year we want to use synthetic biology to development a new strategy transcending these limitations, which needs integrate lead accumulation in earthworm intestine and further permanent treatment.

Inspiration

Considering the earthworm’s intestine is good culture medium for bacteria, we combine bioremediation and microbial remediation together to realize advantageous complementarities. Thus, we chose the dominant symbiotic bacterium in earthworms' intestine-Bacillus subtilis as our engineered bacteria.

To achieve permanent treatment, we decide to transform lead ions into a stable mineral. Lead in soil can form exceptionally stable pyromorphite (Pb₅(PO₄)₃Cl) in the presence of enough soluble phosphate with a Ksp of about 10^-60 - 10^-85, which has passed the EPA standard of US and is unable to extract effective lead through the TCLP method.

While apply in the real world, our engineered bacteria may release to the natural environment. Considering about bio-safety, we also need a suicide module to make our project more practical.

Strategy

Combine all inspirations together, we provide the follow surprising strategy. We plan to use earthworms as the mobile carrier of engineered bacteria and the whole strategy can be divided into three phases:

• In laboratory, engineered bacteria successfully cultured.
• In earthworms' intestines after intaking soil, engineered bacteria expressed phytase forming pyromorphite.
• In external aerobic environment by discharging along with earthworms' intestinal excrement, engineered bacteria committed suicide.

Functional Modules

Phosphate-solubilizing module

In order to form pyromorphite, the neutral phytase gene phy (ycD) from Bacillus subtilis YCD was selected. Phytase can hydrolyze phytic acid or phytate to produce soluble phosphate.

Kill-switch module

Considering of bio-safety, we employed MazF as our suicide protein, which can cleave a single strand of mRNA at a specific sequence site and cause cell death. This is regulated by two factors — CΙ repressor and trigger RNA to confirm specific expression and reduce leakage.

Oxygen-regulated module

The global regulator Nitrate Reductase Regulator (FNR) controls genes that help to promote adaptive growth under oxygen limitation. Promoter nar is activated by oxygen-free via the FNR. The engineered bacteria can "distinguish" the anaerobic environment in the intestine from the aerobic environment in vitro relying on the promoter nar.

Gene circuit

We assembled all the parts and constructed the gene circuit, our engineered bacteria would go through three stages. We hope they can perform functions effectively in three different stages.

Phytase

Our project aims to secrete phytase to immobilize lead ions, so we carry out the following experiments to ensure that our system can secrete phytase properly.

Fig.1.Design of phytase

Purification and validation

Phytase expression and purification

• Centrifugate and collect the supernatant.
• Ni-NAT affinity chromatography.
• SDS-PAGE gel electrophoresis.

Fig.2.Western blot result

Verification of the effect of hydrolysate on lead fixation by enzymolysis and X-Ray Diffraction

Fig.3.Phytase validation

Fig.4.XRD result

• Set up above four groups of experiments.
• Measure changes of lead irons concentration.
• Carry out X-Ray Diffraction.

Kill Switch

To verify the effectiveness of kill switch, we utilize simulation experiment and KS model.

Fig.1. Design of Kill-switch

Experiment

• Construct plasmid of kill switch module.
• Set up the test and control group.
• Transfer the test group to an anaerobic induced environment after culturing engineered bacteria for 12 hours.
• Measure OD₆₀₀ of bacteria liquid by enzyme marker.

Fig.2. OD₆₀₀ of control and test group

Model

To further verify the project, we had also established Kill Switch Model to ensure the effectiveness of kill switch.

The inhibitory effect of CI protein on MazF follows the inhibitory rules

$$\varepsilon_{1}=\left( \frac{\left[ O_{CI-1f}\right]}{ O_{CI-1}} \right) \left( \frac{\left [ O_{CI-2f}\right ]}{ O_{CI-2}}\right)$$

The combination of Trigger RNA and Switch RNA follows the combination principle

$$ K_{1}=\frac{\left [ tr.tsRNA\right ]}{\left [ trRNA\right ]\left [ tsRNA\right ]} $$

• Since CI protein was the key on the expression of MazF, we used ordinary differential equations to simulated various combinations of CI protein RBS and degradation tag added to CI (Fig.13)based on the above rules.
• And then we gave the reasonable combination : B0029 and LVA degradation tag. Under this combination, the concentration of MazF in the intestine was less than the threshold, almost at zero and that out of the intestine was more than the threshold(Fig.14). Therefore, we could ensure the pathway work.

Fig.13. RBS and Degradation tag combination

Fig.14. MazF concentration under our choice

Future Steps

Optimize the experimental plan

For instance, apply flow cytometry or spread plate method to detect the number of living bacteria.

Add the colonization part

Make sure that the engineered bacteria can colonize in the intestine of earthworm for a long time.

Carry out further soil experiments

• Further verify the effect of engineered bacteria under real soil conditions.
• Carry out earthworm experiments under approval of the laboratory and safety policies.

Find and choose more effective parts

Search for more suitable enzyme, more sensitive oxygen-free inducible promoter and switches that have lower basic expression.

Implementation

We envision that our project can ultimately help agricultural producers and we chose cultivated land in southern China, whose lead content is between the risk screening value and the risk control value.
We hope to realize an effective cycle from the laboratory and to the enterprise to the farmer in the real world to achieve the maximum effectiveness of the project.

The laboratory will be mainly responsible for providing technical support.

The biological company will be responsible for the expansion and cultivation of bacteria and the cultivation of earthworms.

Farmers will be mainly responsible for the inoculation of earthworms and application feedback.

Cellular Automaton Model

We had established Cellular Automaton Model to help farmers determine the earthworm releasing strategy. We simulated the effect of various earthworm releasing strategies (Fig.1) according to the evolution rules we set. Based on the results, we suggested that the releasing strategy is:

Put 7500 earthworms into each pile at an interval of 10 meters, and run it twice, each time for three months.

Under the strategy, the lead concentration would reduce by 60% after 6 months(Fig.2), reaching the standard level.

$$ p\left ( i,j \right )= \alpha \times attract\left ( i,j \right )\times \frac{1}{\frac{now_{ew}\left ( i,j \right )}{sum_{ew}}+1} $$ $$ \Delta Pb\left ( i,j \right )= m_{eat}\times \rho_{Pb}\left ( i,j \right )\times now_{ew}\left ( i,j \right ) $$

Fig.1. The effect under various earthworms releasing strategies within 3 months.

Fig.2. The reduction of Pb within 6 months

Contribution to iGEM

BBa_K525515：The LC-MS / MS method

Supply BBa_K525515 with the LC-MS / MS method and related data of bisphenol A. It has the advantages of strong qualitative ability, strong stability and high sensitivity.

Compound	Linear equation	Correlation coefficient	LOD（μg/L）	LOQ（μg/L）
BPS	y=3.11×106x-4.4×105	0.9999	0.002	0.007
BPF	y=5.22×104x-5.2×104	0.9974	0.05	0.17
BPA	y=1.87×104x+1.64×104	0.9996	0.15	0.50

Table1.Linear equation, correlation coefficient, LOD and LOQ of BPS, BPF and BPA in pure water

BBa_K1067006：The A/O -SBR method

Use the A/O -SBR method to improve the problem of BBa_K1067006 that N₂O couldn’t be detected precisely during the denitrification reaction.

Fig.1.(N₂O emission rates and N₂O cumulative emission in A/O- SBR system)

BBa_K1096001：The CRISPRi technology

Supply the data of the influence of knocking down mazEF gene and hipA gene in EcN1917 by CRISPRi on the formation of biofilm and persister bacteria.

Fig.2. The killing curve on effect of mazEF and hipA on the formation of persiser cell

3D Model of BBa_E1010

Build the 3D Model of BBa_E1010 with SWISS MODEL to simulate the true tertiary structure of protein.

Optimize the codon of CⅠand DpnI

Optimize the CⅠ on ThermoFisher SCIENTIFIC and optimize the DpnI by using the method of synthetic biology to use it better in our engineered bacteria.

Human Practices

After interviewing a peasant, an official in Environmental Protection Bureau and an agricultural enterpriser, we knew that heavy metal pollution not only causes losses to ECOMOMY, but also poses a threat to Human Health. In order to promote harmonious development between human and nature, we utilized synthetic biology to remedy lead contaminated soil. SLIM was born.

To let SLIM thrive, we conducted our work in a multi-dimensional way from the health of human, animals，environment and agricultural. Therefore，we consulted related experts and professors, based on whose advice we gradually improved our project.

Dr. Joseph Bressler

“Water companies often add soluble phosphate to prevent lead from flowing into drinking water. Then the key point for you is to know whether there is enough soluble phosphate in the soil.”

“There’s little soluble phosphate but much insoluble phosphorus in soil, I think you could convert insoluble phosphorus into soluble phosphate by enzyme. ”

Professor Guo Xianhua

Professor Li Zhen

“Before choosing the enzyme, it’s important to consider physical and chemical properties of the environment.”

SLIM has grown up! We distributed a questionnaire on people’s attitude to SLIM and their acceptance of SLIM. We found that most people were concerned about food safety but knew little about heavy-metal contaminated vegetables, meaning that we need to greatly enhance public EDUCATION. If they could know more about these vegetables and would like to pay a bit more to buy them, farmers and peasants could benefit more.

We planned to solve an environmental problem at first. One Health inspired us that we can do much more than that, because people, animals and environment are closely connected. We can protect food safety and human health while solving the environmental problem. Farmers and peasants, the biggest stakeholder, can earn more income. Agricultural economy can be promoted. SLIM has been influencing the world and the world has been influencing SLIM.

Science Communication

Science Communication, as the fertilizer, nourishes SLIM to be stronger. We break the boundaries between SCIENCE, EDUCATION and ART, to raise people's environmental awareness, popularizing synthetic biology among people.

Compose a song——"To save the earth, To love my world”

With the rap, we will show our acquaintance with iGEM, expressing our team's firm belief, as well as the inner appeal.

Draw a comic——“A Prophet Came to a Village”

Science picture books in the form of comics can help children learn to actively observe the surrounding environment and find solutions to problems, which lays a good foundation for their future development.

Publish a monthly magazine

It is hoped that middle school students can gain scientific knowledge while reading paper journals and understand the seriousness of soil heavy metal pollution.

Hold NAU-IGEM Campus Competition

Such a campus competition lets students thoroughly comprehend the field of synthetic biology, and understand deeply about synthetic biology on the previous cognition of the social influence and role, experiencing a model contest similar to the iGEM.

Hold an online campaign

We took this educational opportunity to open the door of synthetic biology to these children who might not have learnt that before.

Hold a Yulan-Road Promotion

Our team members delivered presentations and explained more details about iGEM and our project on Yulan-Road.

Acknowledgements and Sponsorship

Acknowledgements

Dr. Bennie I. Osburn
Christie Marie Brunner
Dr. Joseph Bressler
Dr. Jorge L. Mazza Rodrigues
Prof.Ningyue Xu
Prof. Zhen Li
Peixiang Zhou
Yuan Xiao
Prof. Yan Huang
PhD Xinwang Yan

Presented by Team: NAU-CHINA 2020

Team members: Guolong Shi, Zhen Cao, Chengzhu Fang, Yingying Han, Yunong Li, Yuting Sun, Shangping Chen, Peng Chen, Jiaxing Sun, Yifei Zhao, Fengzhi Yuan, Yu Cao, Xiuli Wang, Ziye Lv, Ruijie Zhang, Tianrui Song, Yucheng Chen, Ran Zhang, Xinyue Jiang, Lijun Fan, Jiamao Yang.

College of life sciences, Nanjing Agricultural University, Nanjing, China.

Sponsor

Nanjing Agricultural University
College of life science
Snapgene
ThermoFisher
Biozenturn

References

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