Team:SZU-China/Demonstration
Demonstration
SZU-China 2019 iGEM team aims to kill Mikania micrantha (M. micrantha) by silencing its essential metabolic genes through RNA interference (RNAi) technology. Several recombinant pET-28a (+) plasmids were constructed independently containing specific sequences that could be transcribed and processed into small interference RNAs (siRNAs) to silence the essential metabolic genes of M. micrantha, and transformed into E. coli. Then, the E. coli underwent self-cracking due to the pH-induced conformation change of refractile body (R-body), and RNAi molecules were released and collected to spray on M. micrantha. After 10 days, the weeds turned brown and became wilted. Moreover, we designed the encapsulation of Micrancide as well as the manufacturing line. Also, we developed a recognition program for our drone to spray on M. micrantha automatically.
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Extracting total RNA from M. micrantha leaves to obtain transcriptome information. To ensure the quality of transcriptome, the total RNA extracted should be intact and undegraded (Fig.1).
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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. Recombinant plasmid was constructed and transformed into 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, leading to unroll the R-body expressed inside E. coli to break its cell membrane. The RNAi molecules were released and 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 of Shenzhen University in China. Then, we cut the whole plants into several segments with two nodes and transplanted them. After they produced 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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qRT-PCR and siRNA Detection. We carried out several molecule-level tests on the treated leaves and got the relative gene expression levels of target metabolic genes (Fig.7, 8). In addition, 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 observation. We observed the morphology of the tested leaves and developed a program to measure the degree of the leaf browning (Fig.10, Video 3). According to the measured results, we plotted the etiolation rate of M. micrantha (Fig.11). Finally, the Micrancide-treated plants were dead as expected (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 testing and confirming the efficiency of synthesized siRNAs on killing M. micrantha, we will manufacture the Micrancide through the manufacturing line that we have 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. Also, we have developed the RNAi nanoparticles synthesis kit and designed the box for it.
Fig.15 The encapsulation of Micrancide and synthesis kit
This year, we visited the experimental fields of the AGIS-CAAS and Beijing Genomics Institute (BGI), and 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
