Poster by team SHSBNU_China 2020
The team members are: Runhan Luo, Zhenyue Wu, Zirui Wang, Siqi Qiao, Qingyu Liu, Moqing Liang, Heyang Jiao, Gongze He, Lutong Zhao, Tianya Ma, Muxin Li, Yuhan Li, Jitong Pan, Zhi Qu, Zhimeng Zhang.
Team:SHSBNU China/Poster
Poster: SHSBNU_China
Strangle Locust Plague at Birth
Poster by team SHSBNU_China 2020
The team members are: Runhan Luo, Zhenyue Wu, Zirui Wang, Siqi Qiao, Qingyu Liu, Moqing Liang, Heyang Jiao, Gongze He, Lutong Zhao, Tianya Ma, Muxin Li, Yuhan Li, Jitong Pan, Zhi Qu, Zhimeng Zhang.
Abstract
Locust plague is a harmful disaster, because gigantic locust swarms can eat crops and cause food crisis. However, the existing prevention measures are expensive and not environmentally friendly. Locust plague is mainly caused by the colonizing locusts, while the scattered locusts need to be initiated by serotonin and guaiacol to form colonized locusts. Therefore, we proposed a design to allow E. coli to express four enzymes to degrade serotonin and guaiacol in the intestines of the locusts. To enhance the degradation efficiency, we introduced a self-lysis system in E. coli to allow the enzymes release to the intestinal lumen of locust, which helps ultimately disturb the swarming behavior of locust and avoid the occurrence of locust plagues. For biosafety, we have also introduced a heat-induced suicide switch, which allows E. coli to commit suicide in response to the summer heat when the plagues are reduced.
Inspiration
Locust plague has been spreading in East Africa since the end of 2019. In February 2020, billions of locusts spread from Africa to the Middle East, further expanding the swarm. As a result of locust swarms, a large amount of food and pasture grass were gnawed away, leading to a serious food crisis for local people.
We investigated several approaches to locust control, the most basic of which include physical control (capture) and chemical control (spraying). These methods are not efficient and not environmentally friendly. Thus, we further define the need to develop efficient and environmentally friendly means of locust control, and aims to protect global food security by creating or improving an approach to locust control through the means of synthetic biology.
We investigated several approaches to locust control, the most basic of which include physical control (capture) and chemical control (spraying). These methods are not efficient and not environmentally friendly. Thus, we further define the need to develop efficient and environmentally friendly means of locust control, and aims to protect global food security by creating or improving an approach to locust control through the means of synthetic biology.
Synchronized Self-lysis Circuit
Figure 1. BBa_K3594002 Quorum Sensing Synchronized Self-Lysis Circuit (left) and BBa_K3594003 Quorum Sensing Fluorescent Reporting Device (right)
We constructed these two quorum sensing parts to make the bacteria commit self-lysis and emit green fluorescent. The LuxR gene contained in the bacteria would combine with AHL produced by bacteria, forming LuxR-AHL complex and activates the working of Plux promotor. Then, the promotor would start the transcription of PhiX174E and sfGFP, leading to the self-lysis and green fluorescence emission.
In addition, we examined the work of synchronized self-lysis circuit by using mathematical models. In the predicted result, there are perfect oscillation effect of bacteria population and enzymes concentration and low concentration of aggregation pheromones concentration. As shown in the graphs below, the top, middle and bottom graphics respectively shows the population of bacteria, the concentration of enzymes and concentration of aggregation pheromones.
Guaiacol and Serotonin Degradation
According to a report in the Journal of Insect Physiology in 1973, guaiacol can effectively promote the aggregation behavior of locusts. Moreover, the microbes in aggregated locust’s gut can also synthesize serotonin molecules. Therefore, in order to degrade serotonin and guaiacol efficiently, we constructed a degradation enzyme system for guaiacol and serotonin.
From an AMB Express article in 2019, we learnt that Cytochrome (CYP) and Ferredoxin Reductase (FR) efficiently degrade guaiacol (shown below).
Serotonin can be catalyzed by monoamine oxidase (MAO) to form 5-HIAL, and then form 5-HTOL under the catalysis of NADPH-dependent aldehyde reductase (ALDR), as the image shown below.
There are two composite plasmids in this system and they are respectively responsible for guaiacol and serotonin degradation (shown above). They are constructed on vector pSB4K5, and LuxR-AHL complex as the activator. BBa_K3594004 is a brand new composite part designed and engineered by our team, which could very efficiently degrade guaiacol.
In order to test the function of the degrading enzyme system in the locust intestine, we made comparison between concentration of aggregation pheromones in the presence of degrading system (top one) and the natural growth of pheromone concentration in locust intestine (bottom one) by constructing mathematical models. From the image shown above, we can see that the degrading effect of enzymes is very obvious.
From an AMB Express article in 2019, we learnt that Cytochrome (CYP) and Ferredoxin Reductase (FR) efficiently degrade guaiacol (shown below).
Serotonin can be catalyzed by monoamine oxidase (MAO) to form 5-HIAL, and then form 5-HTOL under the catalysis of NADPH-dependent aldehyde reductase (ALDR), as the image shown below.
There are two composite plasmids in this system and they are respectively responsible for guaiacol and serotonin degradation (shown above). They are constructed on vector pSB4K5, and LuxR-AHL complex as the activator. BBa_K3594004 is a brand new composite part designed and engineered by our team, which could very efficiently degrade guaiacol.
In order to test the function of the degrading enzyme system in the locust intestine, we made comparison between concentration of aggregation pheromones in the presence of degrading system (top one) and the natural growth of pheromone concentration in locust intestine (bottom one) by constructing mathematical models. From the image shown above, we can see that the degrading effect of enzymes is very obvious.
Heat-responsive Suicide Switch
We referred to the achievement of Jilin_China 2018 team. They designed a synthetic heat-inducible RNA-based thermal sensor that is considerably simpler than naturally occurring thermal sensors and can be exploited as convenient on/off switches of gene expression (shown below).
In our project, these two parts (shown below) are designed to let the bacteria commit suicide in hot summer.
When the temperature is lower than 37 ℃, the RBS is hidden, thus the mRNA cannot be translated into amino acids. When the temperature is higher than 37℃, the hairpin structure of the thermal sensor is unlocked, therefore the ribosome could translate mRNA into amino acids, resulting in the fluorescent and lysis effect.
Methodology
Function Verification of Guaiacol and Serotonin Degrading Enzyme System
In order to complete the degradation enzyme system for guaiacol and serotonin, we constructed two plasmids pSB4K5_pTac_CYP_FR (PSHS4) and pSB4K5_pTac_MAO_ALDR (PSHS5) by molecular cloning. We transferred these two plasmids into E.coli BL21, separately. And the following procedures are shown in the picture below.
Mathematical Modeling Verify the function of SLC
In order to secrete enzymes into the locust's lumen efficiently, we introduce the synchronized self-lysis circuit (SLC). This system can promote the interaction between enzymes and substrates in locust’s lumen and improve the degradation effect.
We assume that the change of protein concentration in the locust's lumen is related to the amount of gene expression, the rate of bacterial lysis, the rate of protein degradation, and the rate of locust intestinal peristalsis. We constructed an equation to describe the change of protein concentration and two equations to predict the change of the concentration of pheromone, the equations are shown below.
Function Verification of Heat-responsive Suicide Switch
In order to test the function of heat-responsive fluorescence switch, each component of bacteria with pSHS1 was divided into 3 biological controls, which were placed at 30℃, 33℃, 37℃, 42℃ with the negative control, positive control and blank control LB, and shaking at 220 rpm. We took samples at 0h, 4h, 8h and 20h respectively (shown in the image below), used a microplate reader for spectrophotometer and fluorescence test, and recorded the data in OD600 and Fluorescence 485/510 (Excitation/Emission).
In the next experiment, each component of PSHS2 was divided into 3 biological controls, which were shaken at 220 rpm at 30℃, 33℃, 37℃, and 42℃. Samples were taken at 0h, 2h, 4h, 8h, and 20h respectively.
In order to complete the degradation enzyme system for guaiacol and serotonin, we constructed two plasmids pSB4K5_pTac_CYP_FR (PSHS4) and pSB4K5_pTac_MAO_ALDR (PSHS5) by molecular cloning. We transferred these two plasmids into E.coli BL21, separately. And the following procedures are shown in the picture below.
Mathematical Modeling Verify the function of SLC
In order to secrete enzymes into the locust's lumen efficiently, we introduce the synchronized self-lysis circuit (SLC). This system can promote the interaction between enzymes and substrates in locust’s lumen and improve the degradation effect.
We assume that the change of protein concentration in the locust's lumen is related to the amount of gene expression, the rate of bacterial lysis, the rate of protein degradation, and the rate of locust intestinal peristalsis. We constructed an equation to describe the change of protein concentration and two equations to predict the change of the concentration of pheromone, the equations are shown below.
Function Verification of Heat-responsive Suicide Switch
In order to test the function of heat-responsive fluorescence switch, each component of bacteria with pSHS1 was divided into 3 biological controls, which were placed at 30℃, 33℃, 37℃, 42℃ with the negative control, positive control and blank control LB, and shaking at 220 rpm. We took samples at 0h, 4h, 8h and 20h respectively (shown in the image below), used a microplate reader for spectrophotometer and fluorescence test, and recorded the data in OD600 and Fluorescence 485/510 (Excitation/Emission).
In the next experiment, each component of PSHS2 was divided into 3 biological controls, which were shaken at 220 rpm at 30℃, 33℃, 37℃, and 42℃. Samples were taken at 0h, 2h, 4h, 8h, and 20h respectively.
Result and Conclusion
Guaiacol and serotonin degrading enzyme system
We can see the result obviously in lane 3 and 4, which can prove that the systems we constructed are successful.
In the future, we would determine the activity of enzymes, and determine the activity of enzymes with the synchronized lysis circuit (SLC) because we use this system to improve the degradation effect.
We used mathematical models to predict the degrading effect of guaiacol and serotonin degrading enzyme system. The predicted results are shown below.
According to the results shown above, we know that the proteins are released by SLC continuously, and we speculated that the concentration of enzymes can keep in an interval steadily, which is really effective for the degradation of serotonin and guaiacol. However, without the function of enzymes in SLC, the concentration of aggregation pheromones grows exponentially and reaches highest soon.
Heat-responsive suicide switch
We tested the fluorescent property of BBa_K3594013 and expressed the data in the figure below.
After we verified the function of K2541001 to sense high temperature, we planned to test if high temperature could induce the lysis process of bacteria with BBa_K3594001, which contains a lysis protein PhiX174E as the downstream of K2541001. In the next experiment, each component of BBa_K3594001 was divided into 3 biological controls, which were shaken at 220 rpm at 30℃, 33℃, 37℃, and 42℃. Samples were taken at 0h, 2h, 4h, 8h, and 20h respectively. We recorded the data in OD600 through microplate reader, and plotted the growth status of BBa_K3594001 at different temperatures over time:
In the 20h, the OD value of the bacteria which is putted in 42°C did not drop significantly as we imagine. Because the temperature in shaking incubator is lower than normal temperature, which may influence the result. Therefore, in the future, we would adjust equipment and measure OD again.
Synchronized self-lysis circuit
In this summer, we did a lot of Gibson assembly experiments to ligate the fragments with PhiX174E. However, we didn’t get one living E. coli colony yet. We thought the reason might be that all the colonies with right sequence of pTac-PhiX174E die due to the leaked expression of pTac promoter. Though we didn’t do the experiments, the clear LB plates verified the lysis function of PhiX174E from the side of the “dead” colonies.
If the construction of self-lysis circuit is successful, we will set up two kind of experiments to test the function of this protein, Figure 5 shows a predicted and ideal result by our imagination.
Figure 1. The analysis of SDS-PAGE of BBa_K3594004 and BBa_K3594008. Lane 1 and 2 are BBa_K3594004 and BBa_K3594008 after overnight culture, and then diluted to M9 and cultured to an OD approximately equal to 0.6. Lane 3 and 4 are BBa_K3594004 and BBa_K3594008 adding IPTG for 3h under 37 ℃.
We can see the result obviously in lane 3 and 4, which can prove that the systems we constructed are successful.
In the future, we would determine the activity of enzymes, and determine the activity of enzymes with the synchronized lysis circuit (SLC) because we use this system to improve the degradation effect.
We used mathematical models to predict the degrading effect of guaiacol and serotonin degrading enzyme system. The predicted results are shown below.
Figure 2. The simulation result of protein concentration in locust’s lumen and the comparison of the concentration of pheromone among with SLC and without it. The first graph shows the dynamics of enzymes. The graph two is the concentration of pheromones in locust with our system and the graph three is without our system.
According to the results shown above, we know that the proteins are released by SLC continuously, and we speculated that the concentration of enzymes can keep in an interval steadily, which is really effective for the degradation of serotonin and guaiacol. However, without the function of enzymes in SLC, the concentration of aggregation pheromones grows exponentially and reaches highest soon.
Heat-responsive suicide switch
We tested the fluorescent property of BBa_K3594013 and expressed the data in the figure below.
Figure 3. Normalized Fluorescence of BBa_K3594013 changes with time at different temperatures. The abscissa represents the time, and the ordinate is the Normalized Fluorescence value. From the image, it can be seen that the Normalized Fluorescence increases with the increase of time. The value of Normalized Fluorescence in 42 ℃ increases most obviously, which proves that the suicide switch is effective at high temperatures.
After we verified the function of K2541001 to sense high temperature, we planned to test if high temperature could induce the lysis process of bacteria with BBa_K3594001, which contains a lysis protein PhiX174E as the downstream of K2541001. In the next experiment, each component of BBa_K3594001 was divided into 3 biological controls, which were shaken at 220 rpm at 30℃, 33℃, 37℃, and 42℃. Samples were taken at 0h, 2h, 4h, 8h, and 20h respectively. We recorded the data in OD600 through microplate reader, and plotted the growth status of BBa_K3594001 at different temperatures over time:
Figure 4. The growth of BBa_K3594001 over time at different temperatures. The abscissa represents time, and the ordinate represents the growth of bacteria. It can be seen from the image that the OD value of the bacteria which is putted in 42°C drops at higher temperatures, which proves that temperature control suicide is effective.
In the 20h, the OD value of the bacteria which is putted in 42°C did not drop significantly as we imagine. Because the temperature in shaking incubator is lower than normal temperature, which may influence the result. Therefore, in the future, we would adjust equipment and measure OD again.
Synchronized self-lysis circuit
In this summer, we did a lot of Gibson assembly experiments to ligate the fragments with PhiX174E. However, we didn’t get one living E. coli colony yet. We thought the reason might be that all the colonies with right sequence of pTac-PhiX174E die due to the leaked expression of pTac promoter. Though we didn’t do the experiments, the clear LB plates verified the lysis function of PhiX174E from the side of the “dead” colonies.
Figure 5. The predicted result of the bacterial expression of self-lysis gene. After the addition of IPTG, the expression of PhiX174E, which is a self-lysis gene, would kill the bacteria, resulting in the failure of bacterial growth. Bacteria can grow normally without induction.
If the construction of self-lysis circuit is successful, we will set up two kind of experiments to test the function of this protein, Figure 5 shows a predicted and ideal result by our imagination.
Future Directions
In the future, we want to improve our project by doing quantitative modeling of the enzyme activity to evaluate the decomposition efficiency and practical value of the program on the target molecule. And we will install all the core components together. We also designed a mobile pesticide sprayer. The main idea is to achieve the automation of pesticide sprayer and the complete liberation of replenishment.
Human Practices
· We interviewed Meixiang Cui, an expert and a grassroot worker on plant pest prevention. She has been engaged in prevent locust plagues for more than 20 years. We knew that locust natural habits and current criteria for controlling locust plagues.
· In order to arouse public attention to the locust plague, we conducted a survey on the street, and many people filled in the questionnaires via QR Code. They were very interested in our project and discussed it with us. Most of them knew little about locust plague, so we made science popularization of locust plague to them. They were shocked to learn that locust plagues happened so fluently in this year.
· We went to Institute of Plant Protection, Chinese Academy of Agricultural Sciences, interviewed Professor Zehua Zhang. He is an expert on pest prevention in the national pasture industry. Professor Zhang's research field is the locust monitoring and prevention technology, which fits our research. we consulted him several questions. He said that breaking down pheromones is a feasible way to prevent locust plagues. The destruction of pheromone has a good effect on long-distance areas. For example, some locusts will swarm, then get across many countries or even whole continent. That will cause widespread famine. And the solitude or gregarious type is determined by density and has nothing to do with the insect age. Therefore, using pheromones is a valid way to achieve our goal.
· For the public education, we focused on teenagers. we had two project presentations in our school and a student camp activity. We introduced background information of locust plague and our project design. Students asked some useful questions, such as how to make sure the E. coli get into the intensities of locust. This question reminded us to construct the whole process of our design, from the production of E. coli to release the E. coli on the leaves of crop.
· In order to arouse public attention to the locust plague, we conducted a survey on the street, and many people filled in the questionnaires via QR Code. They were very interested in our project and discussed it with us. Most of them knew little about locust plague, so we made science popularization of locust plague to them. They were shocked to learn that locust plagues happened so fluently in this year.
· We went to Institute of Plant Protection, Chinese Academy of Agricultural Sciences, interviewed Professor Zehua Zhang. He is an expert on pest prevention in the national pasture industry. Professor Zhang's research field is the locust monitoring and prevention technology, which fits our research. we consulted him several questions. He said that breaking down pheromones is a feasible way to prevent locust plagues. The destruction of pheromone has a good effect on long-distance areas. For example, some locusts will swarm, then get across many countries or even whole continent. That will cause widespread famine. And the solitude or gregarious type is determined by density and has nothing to do with the insect age. Therefore, using pheromones is a valid way to achieve our goal.
· For the public education, we focused on teenagers. we had two project presentations in our school and a student camp activity. We introduced background information of locust plague and our project design. Students asked some useful questions, such as how to make sure the E. coli get into the intensities of locust. This question reminded us to construct the whole process of our design, from the production of E. coli to release the E. coli on the leaves of crop.
References and Acknowledgement
References
[1]. M. L. Anstey et al., Science. 323, 627-629 (2009)
[2]. D. J. Nolte et al., Journal of Insect Physiology. 19, 1547-1554 (1973)
[3]. B. Torto et al., Journal of Chemical Ecology. 20, 1749-1762 (1994)
[4]. D. Obeng-Ofori et al., Journal of Chemical Ecology. 20, 2077-2087 (1994)
[5]. S. Fuzeau-Braesch et al., Journal of Chemical Ecology. 14, 1023-1033 (1988)
[6]. R. J. Dillon et al., Nature. 403, 851 (2000)
[7]. J. Nemcsk et al., Neurochemistry International. 9 (4), 505-510 (1986)
[8]. M. O. Din et al., Nature. 536, 81-85 (2016)
[9]. J. García-Hidalgo et al., AMB Express. 9 (34) (2019)
[10]. M. Bortolato et al., Handbook of Behavioral Neurobiology of Serotonin. Chapter 2.4, 203-218 (2010)
Acknowledgement
*Cheng Li and Boyan Ma for giving our team many useful advice;
*Professor Zhang for providing us current situation and background information of locust plague;
*Noposion Agrochemical Company for giving us information of chemical pesticides;
*Bluepha Lab for giving us training, guidance, and lab support;
*MathWorks for sponsoring modeling software.
[1]. M. L. Anstey et al., Science. 323, 627-629 (2009)
[2]. D. J. Nolte et al., Journal of Insect Physiology. 19, 1547-1554 (1973)
[3]. B. Torto et al., Journal of Chemical Ecology. 20, 1749-1762 (1994)
[4]. D. Obeng-Ofori et al., Journal of Chemical Ecology. 20, 2077-2087 (1994)
[5]. S. Fuzeau-Braesch et al., Journal of Chemical Ecology. 14, 1023-1033 (1988)
[6]. R. J. Dillon et al., Nature. 403, 851 (2000)
[7]. J. Nemcsk et al., Neurochemistry International. 9 (4), 505-510 (1986)
[8]. M. O. Din et al., Nature. 536, 81-85 (2016)
[9]. J. García-Hidalgo et al., AMB Express. 9 (34) (2019)
[10]. M. Bortolato et al., Handbook of Behavioral Neurobiology of Serotonin. Chapter 2.4, 203-218 (2010)
Acknowledgement
*Cheng Li and Boyan Ma for giving our team many useful advice;
*Professor Zhang for providing us current situation and background information of locust plague;
*Noposion Agrochemical Company for giving us information of chemical pesticides;
*Bluepha Lab for giving us training, guidance, and lab support;
*MathWorks for sponsoring modeling software.