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Description

Background

Desertification is a natural (unnatural) phenomenon caused by lack of rain, vegetation destruction, overgrazing, wind erosion, water erosion, soil salinization and other factors. The term was first coined in the 1960s and 1970s in the Sahara Region, where it was popularised by years of severe drought that caused unprecedented devastation.

Nowadays, desertification has become one of the most serious environmental problems with local and global effects in the world, and desertified land exists in every continent except Antarctica [1].According to the United Nations, currently desertification has affected one third of the world’s land and one fifth of the world’s population.

Between 1982 and 2015, 6% of the world’s drylands suffered from desertification, and man-made climate change has degraded 12.6% (5.43 million square kilometers) of drylands [2], which is expanding at a rate of 60,000 km2 per year.This causes direct economic losses of up to us $42.3 billion every year, affecting more than 900 million people in more than 100 countries [3,4].

The European Joint Research Center published the World Desertification Atlas (third edition) in 2018 [5].The results show that population growth and changes in consumption patterns are putting unprecedented pressure on natural resources, with more than 75 per cent of the earth’s land area already degraded and projected to be more than 90 per cent degraded by 2050, with nearly 700 million people displaced, with Africa and Asia most affected. Land degradation is a global problem affecting all regions of the world.

[1] Zhang Yongmin, Zhao Shidong. Global Desertification Status and Countermeasures [J]. Western Development,2016(05):17-19.

[2]Burrell, A.L., Evans, J.P. & De Kauwe, M.G. Anthropogenic climate change has driven over 5 million km2 of drylands towards desertification. Nat Commun 11, 3853 (2020).

[3] Li Xing. Current Situation of Desertification in the World and Countermeasures [J]. World Forestry Research,2000(05):1-6.

[4] Farmers. Hazards of Desertification [J]. Green China,2017(09):66-70.

[5]Cherlet, M.; Hutchinson, C.; Reynolds, J.; Hill, J.; Sommer, S.; von Maltitz, G. World Atlas of Desertification; Publication Office of the European Union: Luxembourg, 2018.

Biological soil crust

Biological soil algae are derived from non-vascular bound plants and microorganisms that live on the surface of the soil. The former include algae and moss, and photosynthesis is the dominant source of local growth of the latter. The latter mainly included bacteria, microfungi, etc., which showed decomposition and metabolism effects in crust [1].They make use of mycelium, false roots and secretions to cement tiny soil particles and atmospheric dust, and form a blanket structure with a thickness of 3 ~ 10 mm to cover the ground, becoming the “system engineer” of the land ecosystem.[2].It can fix nitrogen and carbon in the atmosphere, increase soil organic matter, and improve soil [3].After 1990s, people found that biological soil crust plays an important role in preventing land degradation, and its research gradually attracted people’s attention. Since then, scholars all over the world have carried out a comprehensive study on the spatial and temporal pattern, biological composition, morphological structure, ecological function and management of biological soil crust [4].

Algal skinning is the initial stage of biological soil skinning succession. It is based on physical and microbial crusts. With the change of time, the microenvironmental conditions are improved, the sand space is filled with atmospheric dust, the extracellular polysaccharides (EPS) secreted by bacteria form a fine and smooth inorganic layer on the surface, and algae appear and multiply rapidly [5,6].The species and quantity ratio of Cyanobacteria in the crust of biological soil were relatively high [7].Algae can significantly improve soil enzyme activity and soil fertility, and promote the development of algae crust into more advanced lichen or moss crust [8,9].

[1] Budel B. Microorganisms of soil surface biological crust. Berlin: Springer-Verlag, 2005:307-323.[2] Li Xinrong, Zhao Yang, Hui Rong, Su Jieqiong, Gao Yanhong. Review on the progress and trend of rehabilitation ecology research in China. Advances in Geographic Science, 2014,33 (11) : 1435-1443.

[3] Bowker MA, Maestre FT, Escolar C. Biological crusts of a model system for studying the relationship between biodiversity and ecosystem function in soil. Soil Biology and Biochemistry, 2010, 42:405-417.

[4] Hou Chunmei, Chi Xiuli, HUANG Aihua, He Haoyu. Quantitative analysis of literature on the development trend of international biological soil crust research. Journal of Ecology, 2014,34 (4) : 1035-1041.

[5] Concostrina-Zubiri L, Huber-Sannwald E, MartinezI. Biological soil husking under disturbance restoration scenarios: Effects of grazing systems on community dynamics. Ecological applications, 2014,24 (7) : 1863-1877.

[6] Zhang Yuanming. Microstructures and early developmental characteristics of desert surface biological soil crust. Science bulletin, 2005,15 (1) : 42-47.

[7] lilin. Study on species diversity and ecology of cyanobacteria in algae crust of Songnen Grassland. Changchun: Ornithology papers of Northeast Normal University, 2014.Li L. Ecology and Diversity of algae and cyanobacteria crusts in Songnen Grassland. Master thesis. Changchun: Northeast Normal University, 2014.

[8] Song Y, Shu Wei, Wang An. Characteristics of soil algae during the initial succession of copper mine dump. Journal of Soil and Sediment, 2014,14 (3) : 577-583.

[9] Kitzing C, Proschold T, Karsten U. Effects of ULTRAVIOLET light on growth, photosynthetic performance and Sun Protection of different populations of Strepttophyta in alpine soil. Microbial ecology, 2014,67 (2) : 327-340.

Application of Synthetic Biological Methods

Our project focuses on soil improvement. We hope to promote soil crust and increase soil nutrition by increasing the production of extracellular polysaccharide of engineering bacteria. Although overexpression of PGMA and galU genes can increase the production of EPS, the escape of engineered bacteria may cause unpredictable biological pollution. Based on this consideration, we designed a suicide pathway regulated by arabinose operon. When the engineering bacteria overflowed into the normal soil, the suicide pathway was opened due to the high content of arabinose in the normal soil, so as to prevent possible biological pollution.

At present, people have the following means for desertification control:

1. Protect the existing vegetation and plant trees.

2. Allocate water resources rationally.

3. Reduce the pressure of population on land.

It is not difficult to find that these measures need to be macro-control, while our project focuses on the micro level. Our engineering bacteria and cyanobacteria are widely distributed in the soil, and we set up suicide pathways. Therefore, it can be considered that our project is eco-friendly. This is what we need to do with synthetic biology.

Inspiration

Increasing Extracellular Polysaccharide Production

Firstly, we found the synthesis pathway of EPS. In Streptococcus thermophilus, the synthesis of extracellular polysaccharides is shown in the figure[1]:

Glucose enters the hexose diphosphate pathway (EMP) or glucose-1-phosphate is synthesized under the catalysis of PGM, and then UDP is synthesized by UDP glucose pyrophosphorylase (galU). UDP glucose can be used as a raw material for the synthesis of EPS.

After consulting the literature, Fredrik levander and his team overexpressed pgmA and galU genes in Streptococcus thermophilus. Finally, they obtained that when galU and pgmA (encoding glucose phosphate mutase (PGM)) were overexpressed, the EPS yield in S. thermophilus increased from 0.17 g / mol to 0.31 g / mol [2]. Both Bacillus subtilis and Streptococcus thermophilus are Gram-positive bacteria. Therefore, we speculate that overexpression of pgmA and galU genes in Bacillus subtilis can also increase the production of extracellular polysaccharide. Therefore, we hope to overexpress pgmA gene and galU gene in Bacillus subtilis to achieve high extracellular polysaccharide production.

Circuit and Suicide Mechanism

Our circuit is divided into two modules: polysaccharide production and suicide circuit.

The production of polysaccharides is controlled by constitutive promoters. pgmA and galU genes will be transcribed and translated all the time.

We chose arabinose operon as suicide switch and MazF as suicide gene. PC promoter is a constitutive promoter, which initiates transcription and translation of AraC protein gene. When PC promoter begins to transcribe, the transcribed AraC protein binds to the PBAD promoter. At this time, AraC protein suppresses the PBAD promoter and subsequent genes cannot be transcribed and translated. [3] The promoter of PBAD needs to meet the conditions of binding AraC protein and arabinose at the same time. Because the content of arabinose in desertification land is very low [4,5], arabinose operon will not open suicide pathway in desertification land. When the Bacillus escapes to other areas, due to the high content of arabinose in other areas, arabinose and AraC protein will form inducible C protein cind and activate PBAD promoter. Then suicide gene MazF will be transcribed and the engineering bacteria started to suicide.

The Work of The Seniors

After determining the general idea of our project, we began to consult relevant literature to improve the specific experimental scheme. The study of Concostrina-Zubiri L et al. [7]showed that EPS secreted by bacteria could bind soil particles and dust to form large soil aggregates, promoting the production of biological soil crust. Subsequently, we referred to the work of Fredrik Levander et al.[2], who achieved high EPS production by overexpressing the genes pgmA and galU, two key enzymes in the EPS synthesis pathway, and the experimental data showed that this method nearly doubled the production of EPS.

The excellent work of our predecessors made our project design go very well, and we followed their ideas to design our own high-yielding extracellular polysaccharide(EPS) genetic circuit. However, we should always consider the safety of bacteria in the natural environment. Research by Liu Chengzhu et al. showed that arabinose content was low in desertification lands, and with the improvement of soil quality, the content of arabinose increased. Therefore, we chose arabinose as a marker of soil quality and designed a suicide switch with the arabinose operon, so that the bacteria could only function in the place where it was needed — desertification lands. Once it escaped the desertification environment, the arabinose operon would turn on the suicide mechanism. Thus, the biosecurity problem is solved.

[1] Kong Linghui, Zhao Linsen, et al. Research progress of extracellular polysaccharide biosynthesis of Streptococcus thermophilus [J]. Journal of Food Safety and Quality Inspection,2019,（2）： 284-290

[2] Levander F，Svensson M，Radstrom P． Enhanced exopolysa chailde production by metabolic engineering of Streptococcus thermophilus[J] Applied Environmental Microbiology，2002，68(2)：784-790．

[3]Sharon Ogden, Dennis Haggerty. The Escherichia coli L-arabinose operon: Binding sites of the regulatory proteins and a mechanism of positive and negative regulation [J] Biochemistry,1980(7) 3346-3350

[4]Liu Chengzhu, Jia Juan, et al. Source and distribution characteristics of neutral sugar in soil [J] Acta Phytoecology, 2019, 43 (4): 284–295

[5]Li Guoqiang, Tan zhuojie et al. Determination of monosaccharide content in soil by ion chromatography [J] Chinese Soil and Fertilizer, 2019,(1)

[6]Wu Li, Chen Xiaoguo, et al. The role of soil microorganisms in the formation of biological crusts and its ecological significance [J] Environmental Science,2014,(3)：1138-1143

Biological soil crusts under disturbance restoration scenarios: effects of grazing systems on community dynamics. Ecological Application, 2014, 24 (7): 1863-1877

Co-culture of Cyanobacteria and Engineering Bacteria

The algal crusts are based on microbial crusts in soil crusts, so we co-culture cyanobacteria with engineering bacteria in order to achieve better results. Among them, extracellular polysaccharide agglomerates small particle soil, filamentous cyanobacteria further entangle small particle soil to increase soil particle size[6], so as to achieve better soil crust effect.

Development Prospect

1.Improving desertification.

This is the original intention of our project. We hope to promote the formation of biological soil crust through the co-culture system of Bacillus subtilis and cyanobacteria, so as to curb the trend of desertification and make the desert become oasis again.

2.Accelerating natural evolution.

Since our engineered bacteria can produce high extracellular polysaccharides and thus accelerate the formation of soil aggregates, they can promote the formation of algal crust, thus accelerating the natural evolution process.

3.Aiding alien migration.

It’s a wild idea. At present, alien migration has become an interesting topic of increasing concern. So, maybe our project can provide some ideas and ideas on how to turn the barren alien soil into a fertile field for life.

Under the Shadow of the COVID-19 Epidemic

This year, we experienced the COVID-19 epidemic. Due to the epidemic, the opening time of our school was delayed and our team members could not communicate face to face. What’s more, we didn’t start the experiment until July. We tried our best to deal with all kinds of difficulties. We used Tencent conference and other online office media for communication and discussion. In addition, during our time of home isolation, we searched papers and designed experiments, which laid a good foundation for the following experiments. It is worth mentioning that we have held a public lecture on synthetic biology online. Our progress has not stopped although the epidemic almost destroy our normal study and life.

Team:XJTU-China/Description

XJTU-China