Abstract
Upon the excessive deforestation, grazing and reclamation of human beings, desertification has been intensified. A natural sand-fixing system, biological soil crusts, was discovered to fight for desertification. But this natural sand fixation strategy always has little effect when facing the aggressive sand. Thus in our project, an engineered Bacillus subtilis was constructed to effectively produce extracellular polysaccharide, the key component of soil crust via introducing different combinations of key enzymes GalU and PGM. An arabinose-regulated suicide switch was also built to initiate suicide once the engineered bacteria release from the desert environment for biosafety. Furthermore, a symbiotic system of engineered Bacillus subtilis and cyanobacteria was developed to form sand fixers alliance, fighting for desertification. Our project is committed to educating the public about the current situation, hazards and solutions of desertification, and to providing a more convenient and effective strategy for desertification control.
Team:XJTU-China/Poster
Sand Fixers Alliance
Background
Documentation
The desertification means the land degradation in arid, semi arid and dry sub-humid areas which is caused by various factors including climate variability and human activity.
Desertification is one of the most serious environmental problems with local and global effects in today's world, which exists in every continent except Antarctica. Desertification has affected one fifth of the world's population and one third of the world's land, resulting in direct economic losses of up to 42.3 billion dollars every year. By 2018, more than 75% of the earth's land area has been degraded. It is estimated that by 2050, more than 90% of the land is likely to deteriorate and nearly 700 million people will lose their homes.
Deficiency of current sand control measures
At present, the main measures to control desertification include mechanical engineering method, chemical sand fixation method, agricultural sand fixation method and biological sand fixation method. However, the existing methods have their own shortcomings that can hardly be overcome, and can not completely solve the problem of desertification.Mechanical engineering measures---- high cost and large quantities
Chemical sand fixation measures----high cost, and gas permeability should be considered when selecting chemical materials
Agricultural sand fixation measures----slow effect and long cycle
√ biological sand fixation measures----the most effective sand fixation measures to fundamentally improve the local ecological environment.
This year, iGEMers of XJTU-China is committed to solving the above-mentioned problems by means of synthetic biology. While exploring better remediation measures, we found such a material with strong potential—biological soil crust(BSC). It is the product of the natural succession of desert land. BSC has ecological hydrological functions and can effectively prevent soil water and wind erosion. It is also a provider of carbon and nitrogen sources in sand and promotes the evolution of desert vegetationHowever, this kind of natural sand fixation system is slow to form, and only after it accumulates to a certain degree can sand fixation be effective. After consulting a large number of literatures, we decided to take the synthesis of BSC as the breakthrough point and take synthetic biology as the means to design a method that can produce soil crust in large quantities and efficiently in a short time.
After investigation, we found that the main microorganisms involved in the formation of BSC are Bacillus subtilis and cyanobacteria. Therefore, in our project, we constructed a new Bacillus subtilis engineering bacteria and introduced different combinations of key enzymes GalU and PGM to efficiently produce extracellular polysaccharides. It is a key component of soil crusts and can effectively form soil aggregates that absorb and retain water. Besides, in order to prevent engineered bacteria from escaping from the expected desert environment. We added an arabinose-regulated suicide switch to initiate suicide. In addition, we have also developed a symbiotic system of engineered Bacillus subtilis and cyanobacteria to form a solid sand-fixing alliance to further control desertification and lay a "green carpet" for it.
Wet Lab
Engineered bacteria with GalU and PGM expression were able to produce EPS efficiently
Inspiration：
To overexpress the extracellular polysaccharide (EPS) via Bacillus subtilis, we managed to seek for the enzymes positively promote the synthesis of EPS in the bacteria, whose result was galU and pgmA
Design：
We searched for two isoenzymes with higher enzyme activity of galU from Bacillus subtilis, Escherichia coli, and three isoenzymes of PGM from Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. We linked these genes and regulated their transcription by the Bacillus subtilis constitutive promoter.
Results:
We have characterized gene expressions in these plasmids at the transcriptional level using RT-qPCR.
We used anthrone-sulfuric acid method to determine the content of polysaccharides in the culture solution for 24 hours.
The results of gene expression at mRNA level and the yield of EPS in engineered strains have demonstrated our engineering success as expected.
To overexpress the extracellular polysaccharide (EPS) via Bacillus subtilis, we managed to seek for the enzymes positively promote the synthesis of EPS in the bacteria, whose result was galU and pgmA
Design：We searched for two isoenzymes with higher enzyme activity of galU from Bacillus subtilis, Escherichia coli, and three isoenzymes of PGM from Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. We linked these genes and regulated their transcription by the Bacillus subtilis constitutive promoter.
Results:We have characterized gene expressions in these plasmids at the transcriptional level using RT-qPCR.
We used anthrone-sulfuric acid method to determine the content of polysaccharides in the culture solution for 24 hours.
The results of gene expression at mRNA level and the yield of EPS in engineered strains have demonstrated our engineering success as expected.An controllable anti-escape system was functional with suicide MazF protein under regulation of are operon.
Inspiration：
Considering biosafety issues, we also designed a suicide switch based on the arabinose operon. The selected gene is nodA( MazF), which encodes EndoA, an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific but frequently occurring sites, namely, UACAU. Thus the reproduction of the bacteria was repressed on account of the deprivation of mRNA and the corresponding protein.
Design： To compare the regulatory effects of Bacillus subtilis and E.coli arabinose operon and test their efficiency, we constructed two verification plasmids respectively with GFP.
Results:
Results showed these two Ara operons are both functional under induction of different concentrations of arabinose, exhibiting low leakage and wide dynamic range. The Ara operon from Bacillus subtilis showed higher fluorescence intensity, proving that it could provide a higher response expression of the toxic protein gene MazF.
Design: We constructed a suicide circuit regulated by the arabinose operon of Bacillus subtilis, and expressed the toxic protein MazF in an environment containing arabinose to execute the suicide of the engineered bacteria.
Results:
Because of COVID-19 and limited time, we failed to fully test the anti-escape system. But we roughly tested the suicide effect with 1.5μmol/L arabinose, and there were no obvious colonies on the plate after 24 hours of culture, which proved the success of our project, to some extent.
Considering biosafety issues, we also designed a suicide switch based on the arabinose operon. The selected gene is nodA( MazF), which encodes EndoA, an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific but frequently occurring sites, namely, UACAU. Thus the reproduction of the bacteria was repressed on account of the deprivation of mRNA and the corresponding protein.
Design： To compare the regulatory effects of Bacillus subtilis and E.coli arabinose operon and test their efficiency, we constructed two verification plasmids respectively with GFP.
Design: We constructed a suicide circuit regulated by the arabinose operon of Bacillus subtilis, and expressed the toxic protein MazF in an environment containing arabinose to execute the suicide of the engineered bacteria.
Results:
Because of COVID-19 and limited time, we failed to fully test the anti-escape system. But we roughly tested the suicide effect with 1.5μmol/L arabinose, and there were no obvious colonies on the plate after 24 hours of culture, which proved the success of our project, to some extent.
Take soil samples in nearby desert environments and detect the content of main nutrients to provide necessary data for modeling
Model
The Growth Model
Using classic growth model, we highlighted the limit of carbon, nitrogen and phosphorus source on bacterial growth.
We have proved that the two microbes can successfully survive in the soil if other conditions are satisfied, and determined the effect of parameter values on the growth curve.
We also found that B. subtilis will produce considerable EPS in the early stage, which proved our introduction of PGM and GalU is very useful.
We have proved that the two microbes can successfully survive in the soil if other conditions are satisfied, and determined the effect of parameter values on the growth curve.
The Suicide Model
We simulated the transport of arabinose into the cell and the expression of toxin mazF.
If the concentration of mRNA decreases obviously, we can infer that suicide is successfully initiated.
By adding a lower concentration of arabinose than that in the soil, the apoptosis would be triggered within 1 hour.By adding a lower concentration of arabinose than that in the soil, the apoptosis would be triggered within 1 hour.
Without arabinose, mazF concentration is much lower than the threshold. So the leakage of expression does not affect the survival of bacteria.
If the concentration of mRNA decreases obviously, we can infer that suicide is successfully initiated.
By adding a lower concentration of arabinose than that in the soil, the apoptosis would be triggered within 1 hour.By adding a lower concentration of arabinose than that in the soil, the apoptosis would be triggered within 1 hour.
Without arabinose, mazF concentration is much lower than the threshold. So the leakage of expression does not affect the survival of bacteria.
The Expansion Model
With the hope of such a pair of microbes will grow to function on a larger scale, we also modeled microbial expansion in the soil plane. B. subtilis and N. sp reproduce themselves, consume nutrients and produce EPS. The osmotic pressure formed by distribution of cells, EPS and nutrients in the soil can take away cells. When the number of cells reaches the carrying capacity, cells’ pushing also helps them expand.
This is a demo of expansion. The color is lighter if there are more cells (/EPS/nutrient) in a square. We can also change the initial distribution or the parameters to see different scenarios of expansion.
This is a demo of expansion. The color is lighter if there are more cells (/EPS/nutrient) in a square. We can also change the initial distribution or the parameters to see different scenarios of expansion.
Hardware
For hardware, we have produced a hardware called DCSC (Desert climate simulation cabinet) which can precisely simulate the desert environment for the cultivation of cyanobacteria and symbiotic system. According to the large temperature difference between day and night in the desert, we use a resistance for heating and three semiconductors for cooling. With programming, the temperature can be controlled at any value between 5 to 50 degrees Celsius. The climate cabinet is equipped with full-spectrum plant growth light for the best illumination. The most coolest thing is that we use ESP8266 to control these electrical parts and sensors to build an IoT platform based on Blinker. Therefore, you can easily connect with our DCSC on mobile phone through Wi-Fi, remote controlling conveniently and monitor state and data in cabinet in real time. There is a upcoming software update with a new function that our DCSC can automatically simulate the climate condition you choose with the real-time weather data from API interface by pressing only one button.
Human Practices
Discussions with professors of desertification control helped us to perfect the establishment of the desert climate simulation incubator and the design of the cost and scope of the project. We contacted students in desert areas to obtained different desert soil samples and tested soil moisture and organic matter and other contents to provide valid data for the modeling group. Through interviews with experts in microbial cultivation and desert control engineering, we have effectively solved the problems encountered in our experiments and have received the help of providing desert experimental bases in the future.
We are also committed to making our project and synthetic biology more widely known. We collected public awareness of desertification, and recorded MOOC videos to popularize science, which also promoted our project. We held three different lectures and a debate on synthetic biology, as well as laboratory science training for students. Besides, we have used XJTU-iGEM's WeChat public account and Bili Bili network platform to enable more people to participate in our projects and benefit from synthetic biology.
Sponsor and Reference
Refernences
Background Ref:[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
[7]Biological soil crusts under disturbance restoration scenarios: effects of grazing systems on community dynamics. Ecological Application, 2014, 24 (7): 1863-1877
Wetlab Ref:
[1] Ling-Hui, K., Lin-Sen, Z., Yong-Jun, X., Hui, Z., & AI Lian-Zhong XIONG Zhi-Qiang (2019, Janurary). Research advance in the exopolysaccharide biosynthesis of Streptococcus thermophilus. Journal of Food Safety and Quality, 10, 284-290.
[2] Levander, F., Svensson, M., & Rådström, P. (2002). Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus. Applied and Environmental Microbiology, 68(2), 784–790. https://doi.org/10.1128/AEM.68.2.784-790.2002
[3] Pellegrini O, Mathy N, Gogos A, Shapiro L, Condon C. The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. Mol Microbiol. 2005 Jun;56(5):1139-48. doi: 10.1111/j.1365-2958.2005.04606.x. PMID: 15882409
[4] Park, Jung-Ho, Yamaguchi, Yoshihiro and Inouye, Masayori(2011), Bacillus subtilis MazF‐bs (EndoA) is a UACAU‐specific mRNA interferase, FEBS Letters, 585, doi: 10.1016/j.febslet.2011.07.008
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