Team:SZU-China/Demonstrate
Demonstration
SZU-China 2019 iGEM team decided to remove M. micrantha by silencing the essential metabolic gene of it through RNA interference (RNAi) technology. Plasmid pET-28a (+) was constructed with a sequence that could transcribe hairpin siRNA to silence essential metabolic gene of M. micrantha and transformed into E. coli. Then, E coli self-cracked due to refractile body (R-body) induced by the changing pH, and RNAi molecules flowed out and were collected to spray on M. micrantha. After 10 days, the weeds turned brown and wilting. Moreover, we designed the encapsulation of Micrancide as well as the manufacturing line. Then, we developed a recognition program for self-assembly drone to spray on M. micrantha automatically.
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Extracting total RNA from M. micrantha leaves to get transcriptome. To ensure the quality of transcriptome, we required that the total RNA was intact and undegraded (Fig.1). -
Using our siRNA sifting program to select and design the siRNA.
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Fig.1 Integrity of extracted RNA from the leaves of M. micrantha
Video 1. Preprocessing of original documents
Video 2. siRNA sifting process
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Transforming and transcribing. We constructed plasmid and transformed into the E. coli to produce the RNAi molecules.
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Inducing E. coli to self-crack. After obtaining enough RNAi molecules, we added 1mM HCl to lower the pH of reaction system to unroll the R-body expressed inside E. coli to break the cell membrane of it and RNAi molecules flowed out and were collected (Fig.3, 4).
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Fig.3 Changing the pH of the reaction system
Fig.4 The unrolled R-body inside E. coli
Fig.2 Shaking and transcribing
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Planting M. micrantha. We first prepared the M. micrantha for experimental tests. All of the plants were collected from Lihu campus, Shenzhen University, China. Then, we cut the whole plants into several segments with two nodes and transplanted. After they grew new roots, we would spray the RNAi-based herbicide on them (Fig.5).
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Spraying on M. micrantha. We sprayed Micrancide on the weed using self-assembled device to test the efficiency of the herbicide. This device could ensure the least loss on the herbicide during testing.
Fig.6 Spraying on M. micrantha using self-assembled device
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RT-qPCR and siRNA Detection. We carried out several molecule-level tests on the treated leaves and got the relative gene expression of target metabolic gene (Fig.7, 8). Moreover, we tested the introduced siRNA inside the leaves using a G-quadruplex DNA-based, label-free and ultrasensitive strategy (Fig. 9).
Fig.7 Real time quantitative fluorescence PCR to test gene expression
Fig.8 The relative gene expression changes of target gene
Fig.9 siRNA Detection
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Morphological changes. We observed the morphological changes of the tested leaves and develop a program to measure the degree of the leaves browning (Fig.10, Video 3). According to the measuring results, we plotted the etiolation rate of M. micrantha (Fig.11). And finally, the Micrancide-treated plants were dead (Fig.12).
Fig.10 Morphological change of leaves
Video 3. Measuring browning degree program
Fig.11 The Etiolation rate of different treatments
Fig.12 The results of Micrancide-treated plants
Fig.5 M. micrantha planting
After finding the useful siRNAs on the M. micrantha, we will manufacture the Micrancide through the manufacturing line we designed (Fig.13).
Fig.13 The manufacturing line of Micrancide
Fig.14 Device of producing a great amount of Micrancide
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.
Fig.15 The encapsulation of Micrancide
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 (Fig.16, video 4).
Fig.16 Application of Micrancide
Video 4. Demo of self-assembled drone with function of identification
