Team:SZU-China/Design
Design
Mikania micrantha , commonly known as mile-a-minute weed, is an extremely fast growing, sprawling, perennial vine (Asteraceae) and one of the top 10 worst weeds in the world. In the late 1980’s and early 1990’s, Mikania micrantha has dispersed to the coastline of Guangdong, China, and brought a great disaster to the local environment [1]:
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Causing significant damage to forests, farmlands and orchards;
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Leading to a loss of genetic and species diversity;
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Declining the soil and food web stability;
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Altering mineral cycling;
The Spread of M. micrantha Endangers the Other Plants
Some studies demonstrated that it possesses many of the biological characteristics associated with successful invasive plant species [2]:
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Germinating early in the growing season and producing prolific numbers of widely dispersal seeds
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Rooting in each vine node to grow into more new plants
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Smothering other plants to get more living space
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Having an ability to survive harsh conditions
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Growing very fast due to its vigorous metabolism
At present, there are three ways to remove Mikania micrantha [1], that is mechanical and manual control, chemical control and natural enemies control. The first way may have undesirable effects on non-target species and the environment and is a long haul, the second one at present has less efficiency and even will leave toxic chemical residues to the environment, and the third one may bring some unpredictable risks to the local environment. (Click Safety to see more)
SZU-China 2019 iGEM team decides to synthesize the Micrancide, an RNAi-based herbicide for Mikania micrantha, to remove the weed by silencing the essential metabolic gene of it through RNA interference (RNAi) technology.
Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules [3]. It involves multiple RNA-protein interactions characterized by four major steps:
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Assembly of siRNA with the RNA-induced silencing complex (RISC)
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Activation of the RISC
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Target recognition
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Target cleavage of mRNA
RNAi is now known as precise, efficient, stable and better than antisense technology for gene suppression. This technology is precise, efficient, stable and better than antisense technology. It has been employed successfully to alter the gene expression in plants for better quality traits [4]. Hence, inspired by these successful examples, we decided to apply RNAi technology to the development of the herbicide for Mikania micrantha.
Micrancide is quite safer than any other chemical or biological technology:
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Target recognition
Micrancide can only target and silence the essential gene of Mikania micrantha and do no harm to other plants. Besides, Spraying the RNAi-based herbicide is convenient and timesaving.
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Micrancide vs Chemical Control
RNAi molecules are not so stable as other chemicals and are easily degradable, so they are eco-friendly (for more information, see the Stability).
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Micrancide vs Natural Enemies Control
Micrancide, unlike transgenic technique, cannot cause permanent changes in the gene expression of plants. Moreover, it is controllable.
First, we need to extract the total RNA from leaves of Mikania micrantha and send it to bio-company to have its transcript. Then, we analyze the transcript and develop the related sifting program of siRNA that is specific to the Mikania micrantha and obtain the specific siRNA targeted on the essential metabolic gene of it (Fig.1).
Fig.1 The selection of siRNA
We designed a sequence containing several regions that could form hairpin siRNAs and considering the difficulty of hairpin structure synthesis, we just designed four different pairs of regions as an example to form four kinds of hairpin siRNAs linking together, which could help us produce different functional siRNAs at one time. Then we can produce siRNAs for different target genes in large quantities at the same time, so that we could spray RNAi-based herbicide together to increase silencing efficiency.
Plasmid pET-28a (+) containing this sequence was constructed and transformed into ht115(DE3) E. coli. The RNAi molecules transcribed in E. coli induced by IPTG were extracted and sprayed on Mikania micrantha as an RNAi-based herbicide. We also transformed the self-cracking mechanism into the E. coli, which would poke hole in the E. coli, causing the inclusion to flow outside (Fig.2).
Fig.2 Synthesis of RNAi molecules
The effectiveness detection contains three parts: qualitative detection of siRNA introduced into the leaves, quantitative detection of gene expression and the apparent morphology record of the leaves (Fig.3). As for the first part, we compared two methods on the siRNA detection, the Northern Blot and a G-quadruplex DNA-based, label-free and ultrasensitive strategy [5] (Click Measurement to see more on the detection). For the second one, we carried out the qRT-PCR to detect the expression level of the target gene. For the third part, we took the photos of the tested leaves every day.
Fig.3 The Effectiveness Detection
To test the specific of Micrancide, we planted Arabidopsis thaliana and spayed Micrancide on it. After 10 days, we found that the Arabidopsis thaliana did not get any influenced (Fig.4). Due to the limitation of time and the difficulty on the experimental culture of other plants, we just use Arabidopsis thaliana to test the specificity. We will do more tests on the specificity before Micrancide being on the market.
Fig.4 Specificity Detection
We simulated the environment in which it was released into the wild to test the stability of the third generation. Moreover, we also tested the storage conditions. (Click Result to see more about the stability)
After finding the useful siRNAs on the Mikania micrantha, we will manufacture the Micrancide through the manufacturing line we designed. At the beginning, input the Basal Medium with 0.3% Trp and glycerol, as well as the cultured E. coli into the fermentation tank, then add 1mM IPTG. The transcribed RNAi molecules will then be transported into the self-cleaning filter, where the concentration of Trp is zero and pH is 3, which can induce the self-cracking mechanism, leading the outflow of the RNAi molecules. The remained bacteria-rich stream and recycled water will flow back to the tank, while the crude RNAi molecules will go into the next tank with 0.1% DEPC, which is used to remove the RNase in case of the RNAi molecules degradation. After high-temperature and high-pressure sterilization for 30 min, the DEPC and the protein can be removed. Then the RNAi molecules will be concentrated inside the freeze vacuum dryer and poured into several bottles and sealed automatically. Finally, the bottles will be packed and sold to the market (Fig.5).
Fig.5 Micrancide Manufacturing
To make our product more convenient to use and not so easy to be degraded, we designed a box for Micrancide and the bottle to hold it. (Click Product to see more)
This year, after we visit the experimental fields of the AGIS-CAAS and Beijing Genomics Institute (BGI), we came up with the idea of using agricultural drone to spray our herbicide. (Click Application to see more)
[1] Zhang, L Y, et al. "Mikania micrantha H. B. K. in China – an overview." Weed Research 44.1(2004).
[2] Li W H, Zhang C B, Jiang H B, et al. Changes in soil microbial community associated with invasion of the exotic weed, Mikania micrantha H.B.K[J]. Plant & Soil, 2006, 281(1/2):309-324.
[3] Kupferschmidt, K. A Lethal Dose of RNA[J]. Science, 2013, 341(6147):732-733.
[4] Saurabh S, Vidyarthi A S, Prasad D. RNA interference: concept to reality in crop improvement[J]. Planta, 2014, 239(3):543-564.
[5] Yan L, Yan Y, Pei L, et al. A G-quadruplex DNA-based, Label-Free and Ultrasensitive Strategy for microRNA Detection[J]. Sci Rep, 2014, 4:7400.
