iGEM 2025 — Team szu-china

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SZU-China
2025 SZU-C
hina 2025

↑

![](https://static.igem.wiki/teams/5588/pagelogo/notebook.webp)

# Notebook

Discovery begins with documentation.

![](https://static.igem.wiki/teams/5588/pagelogo/notebook.webp)

### Notebook

* [Overview](#section1)
* [Wet lab](#section2)
  + [First-generation RNA](#section2-1)
  + [Second-generation RNA](#section2-2)
  + [Third-generation RNA](#section2-3)
  + [Fourth-generation RNA](#section2-4)
  + [*Metarhizium anisopliae*](#section2-5)
  + [VLP](#section2-6)
  + [*Bacillus subtilis*](#section2-7)
* [Dry lab](#section3)
  + [Modeling](#section3-1)
  + [Software](#section3-2)
  + [Hardware](#section3-3)
* [Product Application Form](#section4)
  + [Trunk Injection Delivery of RNAi Products](#section4-1)
  + [*Metarhizium* Product Design](#section4-2)
  + [*Bacillus subtilis* Product Design](#section4-3)
* [Human Practices](#section5)
* [Wiki](#section6)
* [Heartfelt Thanks](#section7)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Overview

**2024.11.25 Teambuilding**

Complete the recruitment and grouping of team members, establish a personnel organizational framework for project advancement, and ensure the orderly implementation of subsequent work.

**2024.11.29-2025.1.13 Experimental skills and literature reading training**

Organize and conduct training on experimental operation skills and literature retrieval & reading methods, improve team members' experimental capabilities and scientific research literacy, and enable members to carry out subsequent experiments and research more efficiently.

**2025.1.16-2025.3.31 Brainstorming**

Team members conducted creative discussions around various fields related to synthetic biology, generating numerous ideas regarding the application of RNA technology, microbial prevention and control, and other aspects, which provided abundant insights into the determination of the project theme.

**2025.3.31 Determine the project**

Based on the integration of brainstorming outcomes and preliminary research, the citrus aphid control project was selected, with the overall objectives and research scope of the project clearly defined.

**2025.3.20-2025.8.16 First-generation RNA**

The first-generation RNA molecules for aphid control were successfully designed and synthesized, which exhibit a certain inhibitory effect on aphids.

**2025.6.15-2025.8.18 Second-generation RNA**

Small segments of different genes were concatenated to form second-generation RNA, and experiments confirmed that its inhibitory effect on aphids is stronger than that of the first-generation.

**2025.8.1-2025.9.10 Third-generation RNA**

The third-generation RNA was obtained by designing the RNA into a hairpin structure and concatenating them to enhance stability, which showed a more significant lethal effect on aphids in experiments.

**2025.8.23-2025.10.3 Forth-generation RNA**

Fourth-generation RNA was developed, and lethal experiments were conducted.

**2025.8.1-2025.9.21 MS2-VLPs**

An MS2-based Virus-Like Particle (VLP) was constructed to encapsulate RNA molecules, providing vector support for the stable delivery of RNA.

**2025.8.7-2025.9.21 dipeptide**

Studies on dipeptides (TAT & GBP3.1) were conducted, and an attempt was made to conjugate the dipeptides to EGFP for proof-of-concept validation.

**2025.7.15-2025.10.2 *Metarhizium anisopliae***

Studies related to *Metarhizium anisopliae* were conducted to prepare for its combination with RNA technology in aphid control, and to explore the prevention and control potential of the integration of microbial and molecular technologies.

**2025.8.1-2025.9.24 engineered *Bacillus subtilis***

Engineered sucrose-responsive *Bacillus subtilis* was designed, and the expression of methyl salicylate was qualitatively analyzed by LC-MS.

**2025.8.22-2025.9.10 Mini Program**

To address the challenge of lacking user-friendly dsRNA design software in previous RNA molecule design, efforts were initiated to develop a dsRNA design applet.

**2025.04.06-2025.10.05 Model**

The model framework was determined, and the background model (population dynamics model), Model 1, Model 2, and Model 3 were completed.

**2025.07.01-2025.10.02 software design**

A statistical method for the efficacy of first-generation RNAi molecules was proposed, and a visual siRNA sorting platform was successfully constructed.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Wet lab

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## First-generation RNA

**2025.3.29 The protocol for in vitro dsRNA production was determined, and target screening was studied.**

Haocheng Li, Jiachun Chen, Yutong Chen —— The team participated in the Nanjing Synthetic Biology Industry Expo and exchanged views with Shanghai Plant Science Biotechnology Co., Ltd on the implementation status of RNA pesticides.

**2025.3.20-2025.4.10 The team carried out RNA target screening work**

Haocheng Li, Shujing Bu —— The team carried out RNA target screening work.

**2025.4.10-2025.5.10 Design of single-target dsRNA and construction of the vector**

Haocheng Li, Shujing Bu, Xiaying Zhou —— After the team identified the RNAi targets, they initiated the design of first-generation RNAi molecules (single-target dsRNA).

**2025.5.10-2025.6.20 In vitro Transcription Experiment of First-Generation RNAi Molecules, Optimization of Transcription Experimental Protocol**

Xiaying Zhou —— In vitro Transcription Experiment of First-Generation RNAi Molecules, Optimization of Transcription Experimental Protocol

**2025.6.20-2025.8.01 Efficiency Verification of Multiple Terminators for First-Generation RNAi Molecules**

Xiaying Zhou —— In the Experiment for Efficiency Verification of Multiple Terminators in First-Generation RNAi Molecules, the terminator with the optimal efficiency was successfully identified.

**2025.5.11-2025.7.20 visited the "Guangdong Key Laboratory of Epigenetics and the Southwest University Laboratory" to learn.**

Shujing Bu, Minwei Chen —— learn aphid rearing methods under the guidance of professional laboratory personnel.

**2025.7.23-2025.8.16 Conducting Lethality and Silencing Experiments of RNAi Molecules**

Shujing Bu, Minwei Chen —— From July 23 to July 30, the formal experimental phase of lethality and silencing of RNAi molecules will commence.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Second-generation RNA

**2025.6.15-2025.7.05 Determine the design direction of second-generation RNAi molecules and construct vectors**

Haocheng Li, Jiachun Chen, Xiaying Zhou —— On July 10th, the team communicated with Chen Ruoyu, which led to the definition of the design direction for second-generation RNAi molecules—integrating multi-target dsRNA to enhance lethal effects and reduce the risk of pest drug resistance, along with the vector construction for second-generation RNAi molecules.

**2025.7.05-2025.7.20 In vitro transcription experiment of second-generation RNAi molecules**

Haocheng Li, Xiaying Zhou, Ziqi Yang, Ruohan Shi —— The second-generation RNAi molecules were successfully transcribed in vitro.

**2025.7.24-2025.8.18 Aphids in vivo experiment of second-generation RNAi molecules**

Shujing Bu, Minwei Chen, Haocheng Li —— Conduct in vivo aphid experiments with second-generation RNAi molecules.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Third-generation RNA

**2025.8.01-2025.8.12 Construction of the third-generation RNAi in vitro production template**

Haocheng Li, Ziqi Yang —— Construct a tri-shRNA synthetic vector

**2025.8.13-2025.8.16 In vitro transcription experiment of third-generation RNAi molecules**

Haocheng Li, Ziqi Yang —— The team completed the in vitro production of third-generation RNAi molecules.

**2025.8.24-2025.9.10 Lethality Experiment with tri-shRNA**

Shujing Bu, Minwei Chen, Haocheng Li —— The team conducted the lethality experiment of tri-shRNA.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Fourth-generation RNA

**2025.8.23-2025.9.07 Determine the design direction of fourth-generation RNAi molecules and construct vectors**

Haocheng Li, Ziqi Yang —— The team determined Design Direction and Vector Construction for Fourth-Generation RNAi Molecules

**2025.9.09-2025.9.18 In vitro transcription experiment of fourth-generation RNAi molecule**

Haocheng Li, Ziqi Yang —— The team completed the in vitro production of forth-generation RNAi molecules.

**2025.9.19-2025.10.03 bi-amiRNA was used for the lethality experiment**

Shujing Bu, Minwei Chen, Haocheng Li —— The team conducted the lethality experiment of bi-amiRNA.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## *Metarhizium anisopliae*

**2025.7.15-8.10 Construction of Metarhizium anisopliae - Modified Shuttle Plasmids**

Haocheng Li, Jiachun Chenng——Construction and Synthesis of Plasmids with PtrpC Promoter-Driven dsRNA Expression Using Recombinant Vector pBar Obtained from Researcher Duo Lai of Shenyang Agricultural University

**2025.8.11-9.11 Agrobacterium-Mediated Transformation of Metarhizium anisopliae**

Jiachun Chenng——Spores of Metarhizium anisopliae were transformed using the Agrobacterium-mediated transformation method, and then the spores were transferred to a selective medium for further screening. The successful transformation of Metarhizium anisopliae was verified by performing genomic PCR and RT-PCR on its mycelia.

**2025.9.12-9.20 The successfully transformed Metarhizium anisopliae was cultured on a large scale for subsequent experiments.**

Jiachun Chenng——Engineered Metarhizium anisopliae that stably expresses dsRNA was obtained through continuous screening and cultivation.

**2025.9.20-10.02 Experiment on Infection of Toxoptera citricida by Engineered Metarhizium anisopliae**

Haocheng Li, Jiachun Chenng——Toxoptera citricida was infected by soaking in a spore suspension of Metarhizium anisopliae, and the virulence of the engineered Metarhizium anisopliae against Toxoptera citricida was confirmed from a phenotypic perspective.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## VLP

**2025.8.1 Proposition and determination of the protective vector**

Haocheng Li, Qizheng Liu—— team communicated with Liu Xuedong regarding the issue of RNA degradation. They decided to add a protective vector to address this problem. Through literature research and analysis, they found that, unlike chemically synthesized polymer nanocarriers, Virus-Like Particles (VLPs) possess excellent biodegradability and biocompatibility. Therefore, they finally determined to use VLPs

**2025.8.1—8.20 Carry out the design work of VLP delivery vectors and the construction of plasmids**

Haocheng Li, Qizheng Liu, Yuxuan Liu ——team carried out the design work of VLP delivery vectors

**2025.8.7-8.20 Design Work of GBP3.1 and TAT, and Plasmid Construction of GBP3.1 and TAT**

Qizheng Liu, Yuxuan Liu ——The team carried out the design work of GBP3.1 and TAT. To address the issue of determining the optimal position of the His tag in the GBP3.1~egfp~TAT fusion protein (and thereby avoid potential interference with the activity of functional peptides), the project, following the suggestion of Senior Fellow Bai Yongsheng, adopted a strategy of constructing two candidate plasmids (GBP3.1~egfp~TAT~His and GBP3.1~egfp~His~TAT) and planned to conduct direct verification through experiments.

**2025.8.21-8.29 Expression and Verification of Recombinant Fusion Protein TAT-egfp-GBP.31，Plasmid Transformation and Verification**

Xiaying Zhou——The teamCarried out plasmid transformation and verification ， the experiment on the expression and verification of the recombinant fusion protein TAT-egfp-GBP.31

**2025.8.24-9.09 Protein Expression of VLPs，Plasmid Transformation and Verification**

Xiaying Zhou, Qizheng Liu, Yuxuan Liu——The team carried out plasmid transformation and verification .Carry out experiments on protein expression, purification and extraction of VLPs

**2025.8.26-8.28**

Xiaying Zhou, Qizheng Liu, Yuxuan Liu——Verify whether exogenous substances can be transported to young leaves and stems through the vascular tissues of tree trunks; verify whether VLPs can be transported to young leaves and stems through the vascular tissues of tree trunks via injection and Western Blot (WB) experiments.

**2025.8.30 - 9.3 Recombinant Fusion Protein TAT-egfp-GBP.31 Parasite Experiment**

Xiaying Zhou —— Feeding Aphids with Recombinant Fusion Protein TAT-egfp-GBP.31 and In Vivo Fluorescence Observation

**2025.9.4 - 9.21 Frozen Section**

Xiaying Zhou—— Embedded, cryosectioned, stained with specific antibodies, and observed under a laser confocal microscope the aphids that had been fed with the recombinant fusion protein TAT-egfp-GBP.31.

**2025.9.9 - 9.18 Characterization of VLPs, Investigation of the Protective Performance of VLP Nanoparticles on amiRNA**

Qizheng Liu, Yuxuan Liu —— conducted TEM observation on the purified VLPs and a complex experiment to verify whether RNA was encapsulated in MS2 VLPs, and performed a time layer experiment on the purified protein using Benzonase Nuclease.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## *Bacillus subtilis*

**2025.08.01-2025.09.03 Proposal for constructing engineered Bacillus subtilis responsive to sucrose concentration; construction of Bacillus subtilis-Escherichia coli shuttle plasmids**

Haocheng Li,Yutong Chen — Discovered that monitoring changes in aphid populations is of great significance for determining pesticide application timing; proposed designing a biosensor based on sucrose (one of the components in citrus aphid honeydew) to reflect aphid population changes. Constructed and synthesized Bacillus subtilis-Escherichia coli shuttle plasmids capable of expressing pchA, pchB, and BSMT1.

**2025.09.06-2025.09.12 Plasmid transformation into Bacillus subtilis**

Yutong Chen— Prepared competent Bacillus subtilis cells and performed transformation with empty plasmids and recombinant plasmids. Plasmids were extracted after amplification, and successful transformation was verified via plasmid PCR and restriction enzyme digestion.

**2025.09.13-2025.09.24 Sucrose induction and determination of methyl salicylate expression level by LC-MS**

Yutong Chen— Induced with different concentrations of sucrose until reaching the same OD value. Established a standard curve for methyl salicylate standard via LC-MS, and based on this, quantified the expression level of methyl salicylate under induction with different concentrations of sucrose.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Dry lab

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Modeling

**2025.04.06-2025.04.12 Confirmation of the Model Framework**

Bingying Zhu – Conducted literature research and initially proposed a modeling framework; discussed with Professor Ailin Zhang on April 11 to confirm the top-level design of the modeling framework; completed the modeling framework.

**2025.04.13-2025.05.02 Completion of the Background Model (Population Dynamics Model)**

Bingying Zhu – 2025.04.13-2025.04.19: Collected annual temperature data of Guangdong Province in 2024, implemented a temperature-growth rate function, and verified that Guangdong is in the suitable temperature range of 15-30°C for 70% of the time; established an aphid population dynamics model based on the Logistic equation, and introduced a temperature-driven term and environmental carrying capacity.

Bingying Zhu – 2025.04.20-2025.04.26: Introduced the Holling Type II functional response to describe natural enemy predation; reviewed literature on the daily predation amount of ladybugs (56-150 individuals) and found that the control threshold cannot be simply derived; proposed a density-dependent S-shaped function for wing morph conversion, and established the local diffusion dynamics of alate aphids.

Bingying Zhu – 2025.04.25: Discussed with Professor Ke Pan and confirmed the introduction of spatial heterogeneity to align with the actual orchard environment.

Bingying Zhu – 2025.04.27-2025.05.02: Established a spatial function of environmental carrying capacity; derived the formula for dynamic control threshold; completed a 20-day simulation of a 5×5 grid orchard using the RK45 algorithm, and generated a population dynamics animation.

**2025.05.03-2025.05.11 Completion of the Aphid Feeding Model (Model 2) and Initiation of Integrating the Monitoring Module (Model 1)**

Bingying Zhu – Conducted literature research on phloem fluid mechanics and confirmed the applicability of the Hagen-Poiseuille equation; modified the equation for the conical structure of aphids' piercing-sucking mouthparts; established a pesticide intake rate model; wrote the code for the feeding model.

**2025.05.12-2025.06.08 Completion of Model 1**

Bingying Zhu – 2025.05.12-2025.05.18: Clarified and constructed the forward modeling of the monitoring mechanism chain.

Bingying Zhu – 2025.05.19-2025.05.25: Conducted inverse modeling to infer the number of aphids from the detected MeSA concentration.

Bingying Zhu – 2025.05.26-2025.06.08: Plotted model curves and conducted parameter sensitivity analysis, etc.

**2025.06.09-2025.09.29 Completion of the Spatiotemporal Distribution Model (Model 2)**

Bingying Zhu – 2025.06.09-2025.07.28: Established the theoretical framework: decomposed three major physical processes; completed the convection-diffusion equation; searched for relevant parameters.

Bingying Zhu – 2025.07.15: Discussed the numerical solution scheme with Professor Ailin Zhang and confirmed the simulation dimension.

Bingying Zhu – 2025.07.29-2025.09.29: Wrote a solver to realize concentration field calculation; generated a spatiotemporal distribution animation.

**2025.08.20-2025.10.5 Completion of Lethal Data Fitting + Multi-Objective Optimization for Model 2**

Bingying Zhu – 2025.08.20-2025.10.4: Lethal effect modeling: the data fitting results for prediction were poor; after communicating with Professor Ailin Zhang, an algorithm suitable for small samples was selected; fitted the RNA pesticide data; generated a 3D lethal surface plot.

Bingying Zhu – 2025.09.3-2025.10.5: Multi-objective optimization framework: defined the decision variable space; constructed a bi-objective function; designed constraint conditions.

Bingying Zhu – 2025.09.22: Discussed the selection of optimization algorithms with Professor Ailin Zhang and confirmed that NSGA-II is suitable for multi-objective non-convex problems.

Bingying Zhu – 2025.09.17-2025.10.5: Implemented the NSGA-II algorithm; solved the Pareto optimal solution set; generated the Pareto frontier curve.

**2025.06.09-2025.09.26 Initiation of Integrating Model 3 and Completion of Model 3**

Bingying Zhu – 2025.06.09-2025.07.05: Lethal mechanism of Metarhizium anisopliae: established a cumulative lethal probability model; combined the infection rate with Michaelis-Menten kinetics and environmental correction.

Bingying Zhu – 2025.07.06-2025.08.01: Spatiotemporal distribution model of spores: dynamic wind field simulation; control equations; multi-physical process modeling; simulation results.

Bingying Zhu – 2025.08.02: Discussed the improvement direction with Professor Mo.

Bingying Zhu – 2025.08.02-2025.08.28: Improved the SIR transmission dynamics.

Bingying Zhu – 2025.08.29-2025.09.26: Conducted multi-objective optimization.

**2025.09.30-2025.10.05 Model Collation**

Bingying Zhu – Summarized the overall framework; organized the parameter table; conducted final review and code release.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Software

### APHiGEM – Fusion dsRNA Computer-Aided Design System

**2025.06.15-2025.06.20 Initial Statistical Program for RNAi Molecule Potency**

Haocheng Li – Designed a statistical program for the design of fusion dsRNA.

**2025.07.07-2025.07.14 Proposal of the New-Generation dsRNA Design System**

Zhuoming Zou – During the team's process of designing dsRNA using existing platforms, it was found that the existing software was very inconvenient to use. Meanwhile, the team had a demand for designing fusion dsRNA, so he proposed the construction of a new-generation fusion dsRNA Computer-Aided Design System.

**2025.07.22-2025.07.31 Construction and Abandonment of the Initial Model**

Zhuoming Zou – Constructed the initial siRNA algorithm model, but after in-depth discussions with team members, loopholes were found in the existing algorithm logic, so we abandoned the initial model.

**2025.08.01-2025.08.07 Development of the Second-Generation siRNA Design System**

Zhuoming Zou – After consulting bioinformatics expert Shaoxiang Wang, he improved the underlying logic of computer-aided siRNA screening and developed the second-generation siRNA design system.

**2025.08.08-2025.08.27 Construction of the Off-Target Database**

Zhuoming Zou – Based on user feedback on software usage, he proposed and constructed the team's own off-target database to address potential biosafety loopholes.

**2025.08.28-2025.09.20 Integration of the Off-Target Analysis Module with the System**

Zhuoming Zou – Expanded the species and sequences in the off-target database, added an off-target analysis module based on the second-generation siRNA design model, and successfully integrated the database with the software.

**2025.09.21-2025.10.02 Completion of Software Engineering and Deployment**

Zhuoming Zou – Developed a large number of detection scripts and extension tools to enhance the software's scalability, containerized the software using Docker, and successfully submitted it to the iGEM official GitLab repository.

### YOLO Visual Recognition

**2025.04.05 Proposal of the Pest and Disease Visual Recognition System**

Zhuoming Zou – During the research at the agricultural material store of Shanwei Suili Agricultural Cooperative, he learned that farmers were unable to accurately identify the pest and disease outbreaks of the current season, which posed significant obstacles to the selection of pesticides and timely pest and disease control. Thus, he proposed developing a visual recognition system to assist farmers in this work.

**2025.04.06-2025.06.12 Extensive Research on Visual Large Models**

Zhuoming Zou – Extensively reviewed various potential visual large models and related literature.

**2025.06.13-2025.07.15 Confirmation of the YOLOv Series Model Scheme**

Zhuoming Zou – Gained inspiration through exchanges with various iGEM teams and decided on the plan to develop the recognition system using YOLOv series visual large models.

**2025.07.16-2025.07.19 Completion of the First Dataset Training for the Model**

Zhuoming Zou – Conducted the first dataset training on a local computer using the pre-trained model provided by the official YOLO platform.

**2025.07.20-2025.08.26 Model Fine-Tuning**

Zhuoming Zou – To improve the model's accuracy, Zhuoming Zou continuously tried various parameter combinations and optimized the dataset.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Hardware

### Aphid Incubator

**2025.05.11**

Team members – Proposal of the Multifunctional Aphid Incubator

**2025.05.12-2025.06.20 Completion of the Preliminary 3D Modeling of the Incubator**

Zhuoming Zou – Based on his understanding of existing biological incubators on the market and in light of the needs for aphid rearing, Zhuoming Zou used 3dMax software to conduct the initial modeling of the incubator.

**2025.06.21-2025.07.02 Proposal of the Double-Layer Water Circulation Temperature Control System**

Zhuoming Zou – To achieve the preset constant temperature function, Zhuoming Zou conducted literature research and proposed the use of a water circulation temperature control system with double-layer incubator walls.

**2025.07.03-2025.07.13 Termination of the Aphid Incubator Project**

Zhuoming Zou – After communicating with the team and consultants, we estimated the cost, difficulty, and actual effectiveness of the project. Finally, we unanimously agreed that the project had high practical operation difficulty and a low possibility of achieving the expected results, so we abandoned this project.

### Laser Minimally Invasive (Technology)

**2025.04.11 Proposal of the UV Laser UAV Minimally Invasive Drug Delivery Scheme**

Zhuoming Zou – During the investigation of aphids' living habits, it was found that aphids are pests that feed on plant sap using piercing-sucking mouthparts. To enable the team's RNA pesticides to enter plants more effectively, Zhuoming Zou proposed using UV lasers combined with UAVs to perform minimally invasive treatment on citrus leaves.

**2025.04.12-2025.06.11 Successful Construction of the UV Laser Minimally Invasive System**

Zhuoming Zou – After extensively reviewing various materials, he constructed a set of UV laser minimally invasive systems based on the principles of common infrared laser marking machines.

**2025.06.12-2025.07.03 Project Obstacles**

Zhuoming Zou – Due to the difficulty in obtaining raw materials for system construction (such as crystals and lasers) and their high costs, we communicated with technical personnel from relevant companies with the help of the lead researchers. Regrettably, we were still unable to resolve these difficulties.

**2025.07.04 Termination of the Project**

Zhuoming Zou – In the team meeting, considering the excessive technical difficulty of the project, we finally abandoned this scheme.

### CitrusShield

**2025.8.20-8.30 Construction of the Basic Sensor Hardware**

Haocheng Li – Used sensor components such as STM32 to build a sensor capable of multi-environmental parameter detection.

**2025.8.31-9.10 Construction of the Mini Program Frontend**

Haocheng Li – Built the frontend interface of the mini program and integrated the community communication function into it.

**2025.9.11-9.20 Cloud Deployment of Sensor Data**

Haocheng Li – Transmitted sensor data to the cloud via WiFi and connected it to the frontend environmental monitoring module of the mini program.

### Trunk Injection-Related Hardware

**2025.8.20-9.18 Trunk Injection Plugs**

Haocheng Li – Designed the first and second generations of trunk injection plugs.

**2025.9.20-9.25 Syringes**

Haocheng Li – Designed the model of the trunk syringe.

**2025.9.20-9.25 Trunk Injection Plugs**

Haocheng Li – Conducted 3D modeling of the trunk injection plugs.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Product Application Form

Our product application forms detail the development and design of various delivery systems and biological control products for citrus pest management.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Trunk Injection Delivery of RNAi Products

**2025.8.10 - 9.30 Trunk Injection Delivery of RNAi Products**

Li Haocheng - Designing the use of trunk injection technology to deliver agents into plants

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## *Metarhizium* Product Design

**2025.8.20 - 8.30 *Metarhizium* Product Design**

Li Haocheng - Designed to deliver *Metarhizium anisopliae* in the form of oil-based spore suspension capsule spraying

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## *Bacillus subtilis* Product Design

**2025.9.20 - 9.30 *Bacillus subtilis* Product Design**

Li Haocheng —— Design of hydrogel-based sustained-release capsules for the delivery of *Bacillus subtilis*

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Human Practices

**2025.2.22 4th iGEM Community Day**

Team members – Participated in the Community Day event, collected feasibility analyses of proposals submitted by various groups and team interest assessments, and selected four projects for in-depth research: Hepatocyte Single-Base Editing for Hepatitis B Treatment, Acne Management, Litchi Preservation, and Citrus Aphid Control.

**2025.2.24 iGEM Experience Exchange**

Yuxuan Liu – Exchanged ideas with Zejia Wei, former team leader of the 2024 iGEM HP team and 2025 iGEM Ambassador, and obtained the suggestion to conduct field research first.

**2025.3.09 Preliminary Field Research**

Yuxuan Liu, Yutong Chen, Minwei Chen – Conducted research in the main citrus-producing areas of Shanwei, Guangdong. They communicated with Manager Wang from the local rural experimental base and 3 farmers, and found that local growers were severely affected by pests. Farmers mainly relied on chemical pesticides for pest control, but faced problems such as high costs, drug resistance, and slow effectiveness. Farmers held a wait-and-see attitude towards biological control and put forward demands for low-cost, efficient, safe, and thorough pest control solutions.

**2025.3.17 Research at Ketang Town Agricultural and Rural Service Center**

Yuxuan Liu, Yutong Chen, Minwei Chen – Conducted research at the Ketang Town Agricultural and Rural Service Center. They communicated with Director Xuehui Wu about local agricultural types, crop sales, pest control methods, and the implementation of projects such as biological control and RNA pesticides. They also introduced the core principles of RNA biopesticides and discussed issues related to reducing pesticide abuse and drug resistance.

**2025.3.21 Expert Exchange**

Yuxuan Liu, Qizheng Liu, Zhuoming Zou, Ruohan Shi – Exchanged ideas with Professor Beixin Mo from Shenzhen University on pest control strategies and the principles of RNAi technology. They confirmed that RNA pesticides have advantages such as species specificity, high efficiency, safety, and ecological friendliness.

**2025.3.29 Nanjing Synthetic Biology Industry Expo**

Yutong Chen, Jiachun Chen, Haocheng Li – Attended the Nanjing Synthetic Biology Industry Expo.

**2025.3.30 Guangdong-Hong Kong-Macao Greater Bay Area HP Salon**

Team members – Participated in the salon event, shared the project proposal with other teams, and collected feedback through exchanges.

**2025.3.31 Visit to BGI Genomics**

Team members – Visited BGI Genomics to learn about RNAi-related R&D and industrialization information, which provided technical references for project design.

**2025.4.05 Field Research in Taohe Town, Haifeng County, Shanwei City**

Team members – Visited agricultural material stores and individual growers, collected information on pesticide usage habits, costs, effectiveness, farmers' demands, and the agricultural material supply chain. They summarized the core demands: low cost, high cost-effectiveness, compatibility with existing application methods, extended control period, and low pesticide residues.

**2025.4.12 Visit to Citrus Deep-Processing Enterprises: Understanding Raw Material and Pesticide Residue Issues**

Team members – Visited citrus deep-processing enterprises. Mr. He, the person in charge, mentioned that poor raw material quality reduces processing quality and increases additive dosage, while excessive pesticide residues require additional processing links, which push up costs.

**2025.4.19 Research at Suili Agricultural Cooperative: Exploring Cooperative Promotion Paths**

Team members – Conducted research at Suili Agricultural Professional Cooperative in Shanwei City, and learned about the cooperative's land contracting and management model. Cooperative members are sensitive to costs and pay attention to the economic benefits of collective promotion, brand enhancement, and member recognition. Cooperatives play an important role in collective procurement, standardized unified pesticide application, risk sharing, and policy implementation demonstration. The team realized that new technologies can only be implemented through promotion via cooperatives. They also learned that traditional chemical pesticides lead to "insect-hole fruits" and pesticide residue issues, which force citrus to be sold at low prices; solving these problems can increase the selling price. The cooperative reported that the initial bead-shaped products had issues such as insufficient application flexibility, high prices, and easy aging and adhesion during storage. The team then iterated the product into an RNAi solution form.

**2025.4.26 Deep Blue Charity Sale Activity: Understanding Consumers' Needs and Perceptions**

Team members – Participated in the Deep Blue Charity Sale Activity and communicated with ordinary consumers. They learned that consumers pay attention to citrus freshness, taste, and cost-effectiveness, and recognize the attributes of being additive-free and low in pesticide residues, but are unwilling to pay extra for related labels. This highlighted the importance of community promotion.

**2025.5.4 Research at Fengze High-Tech Agriculture: Understanding Enterprise-Level Pest Control Demands**

Team members – Conducted research at Shanwei Fengze High-Tech Agricultural Co., Ltd. (Chikeng Town) and communicated with Mr. Guo Xu (the company's owner) and Jiantao Wei (First-Class Section Member of Chikeng Town Public Service Office, Haifeng County). They learned that most large enterprises outsource pest control to spraying service companies, and enterprises have high requirements for product quality, price, and after-sales service. Cooperating with planting bases can help obtain professional feedback to guide product improvement and promotion.

**2025.5.10 Visit to DJI and Tianwei Agriculture: Research on UAV Pesticide Application**

Team members – Visited DJI and Shanwei Tianwei Agricultural Technology Co., Ltd. (a spraying service company). Huafeng Lin, the person in charge, introduced that 50%-60% of planting areas in the South China production region currently use UAVs for pesticide application, which requires higher fluidity and dispersibility of pesticide formulations.

On the same day, the team participated in the 1st Exchange Meeting of the SUSTech-SynBio Community.

**2025.5.11 Insect Rearing Experiments and Pesticide Supplier Research**

Team members – Visited large-scale pesticide suppliers and communicated with Mr. Wang, the person in charge. They learned that suppliers attach more importance to the cost-effectiveness of new pesticides, have their own distribution networks, and need to demonstrate the actual effectiveness of products to customers.

**2025.5.16 Research at Fresh Fruit Purchasers: Confirming the Market Value of Low-Pesticide-Residue Products**

Team members – Conducted research at the local largest fresh fruit purchaser and communicated with Lisheng Chen, the purchasing supervisor. They learned that excessive pesticide residues or poor citrus quality will affect access to large supermarkets and export markets, leading to a significant decline in profits. This clarified the core goal of the project: developing efficient insecticidal RNA pesticides with low residues to help the industrial chain enhance added value and market competitiveness.

**2025.5.17 Participation in South China Exchange Meeting**

Team members – Participated in the 9th iGEM South China Regional Meeting.

**2025.5.20 Consulting Entrepreneurship Mentor: Discussing Project Implementation and Patent Protection**

Team members – Consulted entrepreneurship mentor Jinxu Wang on the implementation of Project Aphigo. The mentor pointed out that the team needs to overcome two major barriers: supply chain stability and pesticide registration certificate qualification. He also reminded the team to lock in core achievements through intellectual property rights. The team then researched the patent layout case of Shanghai Zhishengyougu Biotechnology Co., Ltd.

**2025.5.21 Participation in Taiziwan Primary School EDU Activity**

Team members – Participated in EDU-related activities at Taiziwan Primary School of Shenzhen Shekou Yucai Education Group.

**2025.6.7 Contacting Test Fields: Promoting the Implementation of Field Experiments**

Team members – Contacted the Longhua Institute of Bio-industry Innovation, Shenzhen University for subsequent experimental test fields. The person in charge led the team to visit existing experimental projects.

**2025.6.8 Participation in EDU Activity at Central Primary School of Shenzhen-Shanwei Cooperation Zone**

Team members – Participated in the Education Activity at the Central Primary School of Shenzhen-Shanwei Cooperation Zone.

**2025.6.13 Consulting Registration Experts: Clarifying the Importance of Pesticide Registration**

Team members – Consulted Ning Xie, an expert from the Pesticide Registration Evaluation Committee, regarding pesticide registration issues.

**2025.6.15 Research at Agricultural Material Stores: Collecting Product Application Feedback**

Team members – Conducted research at Lihong Agricultural Materials Store, Jinshi Town, Chao'an District, Chaozhou City. Mr. Lin, the store owner, reported that he attaches more importance to product safety and ease of use, and hopes that products can solve pest problems to ensure citrus yield.

**2025.6.21 Visit to Agricultural Science and Technology Center: Discussing the Feasibility of RNA Pesticide Implementation**

Team members – Visited the Chaozhou Agricultural Science and Technology Development Center and communicated with Director Liao.

**2025.6.28 Visit to Agricultural Technology Extension Center: Understanding Product Pilot and Promotion Mechanisms**

Team members – Visited the Agricultural Technology Extension Center and communicated with Ms. Fu, a staff member.

**2025.7.10-7.11 Hosting "Little Migratory Bird Summer Camp and Family Open Day" Activity**

Team members – Hosted the "Little Migratory Bird Summer Camp and Family Open Day" activity, inviting children and their parents to visit the iGEM Laboratory.

**2025.7.14-7.16 Participation in iGBA Activity**

Yuxuan Liu, Ruohan Shi, Xinyue Feng, Zhuoming Zou – Participated in the 3-day iGBA Activity.

**2025.7.15 Research on Shenzhen Synthetic Biology Research Infrastructure**

Yuxuan Liu, Ruohan Shi, Xinyue Feng, Zhuoming Zou – Conducted research on Shenzhen Synthetic Biology Research Infrastructure.

**2025.7.17 Participation in Blue Crystal Online Exchange Event**

Yuxuan Liu, Ruohan Shi, Minwei Chen – Participated in the Blue Crystal Online Exchange Event.

**2025.7.26-7.27 Participation in iGEM Startups Summer School**

Yuxuan Liu – Participated in the iGEM Startups Summer School.

**2025.7.27 Participation in Community Education Activity: Theme of "Clothing, Food, Housing and Transportation"**

Yuxuan Liu, Qizheng Liu, Minwei Chen – Participated in the Community Education Activity with the theme of "Clothing, Food, Housing and Transportation".

**2025.7.30 Research at Raoping Cooperative: Discussing Promotion and Online Sales**

Team members – Conducted research at Shanmeixiang Rice Professional Cooperative in Raoping County and communicated with Shujie Chen. They learned about local agricultural product procurement and sales methods, visited the sorting and packaging process, and discussed ways to improve online sales channels.

**2025.8.6 Visit to Jingtai Technology: Strengthening Supply Chain and Quality System Construction**

Team members – Visited Jingtai Technology for exchanges and learned about the importance of raw material stability and supply continuity in intelligent production. The team plans to add alternative raw material suppliers, establish a full-process quality inspection system, and expand the production capacity of contract manufacturers to avoid supply chain risks.

**2025.8.6-8.8 CCiC**

Team members – Participated in the CCiC activity.

**2025.8.9 BASIS Visited Shenzhen University Team**

Team members – The BASIS visited the SZU-China.

**2025.8.11 Hosting International Youth Study Tour and "Liyuan Trip" Summer Camp**

Team members – Hosted the International Youth Study Tour and "Liyuan Trip" Summer Camp, led the participants to visit the iGEM Laboratory of Shenzhen University, and explained the project to them.

**2025.8.23 Return Visit to Longhua Institute of Bio-industry Innovation: Promoting Cooperation on Test Platforms**

Team members – Made a return visit to Mr. Zheng from the Longhua Institute of Bio-industry Innovation, Shenzhen University. Mr. Zheng stated that he is willing to support the subsequent optimization experiments of Project Aphigo (e.g., formulations, application methods) and hopes to jointly build China's first standardized RNA pesticide testing platform.

**2025.8.28 Exchange with BWYA Team**

Team members – Exchanged ideas with the BWYA Team.

**2025.8.29 Shenzhen Popular Science Education Federation Visited the iGEM Laboratory**

Team members – The Shenzhen Popular Science Education Federation visited the iGEM Laboratory.

**2025.9.1 Compiling the Popular Science Manual Debunking Myths about Synthetic Biology**

Yuxuan Liu – Participated in compiling the popular science manual Debunking Myths about Synthetic Biology.

**2025.9.2-9.5 Conducting Campus Recruitment Promotion Activities**

Team members – Conducted 3 large-scale campus recruitment promotion activities at the School of Life Sciences, Medical College, and Advanced Research Institute of Shenzhen University, respectively, explaining synthetic biology and iGEM-related content to the participants.

**2025.9.6 Follow-Up Visit to Individual Growers**

Team members – Conducted a follow-up visit to individual citrus growers.

**2025.9.7 Participation in Community Activity at Nanshan District Party-Mass Service Center**

Team members – Participated in a community activity at the Nanshan District Party-Mass Service Center.

**2025.9.9 Conducting SDGs Cooperation with iGEM VIT Vellore**

Team members – Conducted SDGs cooperation with the iGEM VIT Vellore.

**2025.9.11 Host an Online Lecture on International Cooperation**

Team members – Host an online lecture on international cooperation.

**2025.9.14 Conduct a Survey on the Retail Market**

Team members – Surveyed citrus retail stores, fruit retailers, supermarkets, and New Year citrus plant vendors.

**2025.9.15 Accept Media Interviews**

Team members – Interviewed by Reporter Gao.

**2025.9.17 Collaborate with the ASU iGEM**

Team members – Collaborated with the ASU iGEM.

**2025.9.21 Follow-up Visit to the Fruit Tree Base in Jinshi Town, Chaozhou: Exchange on Field Application Feedback**

Team members – Visited Lao Wang Fruit Tree Base in Jinshi Town, Chaozhou, and communicated with the person in charge, Wang Fude.

**2025.9.24 Launched the Collaborative Project "The Do-Not List for the iGEM Competition"**

Team members – Launched the collaborative project "The Do-Not List for the iGEM Competition".

**2025.9.27 Participated in the Shenzhen University Anniversary Carnival**

Team members – Participated in the 2025 Shenzhen University Centennial Carnival.

**2025.9.30 Jointly Display Posters with the SMBU**

Team members – Jointly displayed the project posters with the SMBU.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Wiki

**2025.5.25-2025.6.24**

Xinyue Feng – Browse a large number of excellent learning-related wiki pages, accumulate inspiration and creativity.

**2025.6.25-2025.7.14**

Xinyue Feng – Familiar with the official iGEM wiki construction rules

**2025.7.15-2025.8.17**

Xinyue Feng – Learning HTML, CSS, JavaScript

**2025.8.18-2025.9.2**

Xinyue Feng – Learning Ps, Ai, Id software and the required special effects

**2025.9.3-2025.9.20**

Xinyue Feng – built the main framework of our wiki webpage.

**2025.9.21-2025.10.6**

Xinyue Feng – completed all the necessary web pages for us one by one.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Sincerely thank

Here, we would like to extend our most sincere gratitude to all parties who have helped the SZU-China 2025 successfully complete the creation of the wiki! We thank the advisors for their valuable guidance, and every team member for their wisdom, hard work, and seamless collaboration. Meanwhile, we also thank the school and laboratory for their support, as well as all those who have provided assistance to us throughout the process. It is everyone's collaborative efforts that have led to the successful creation of this wiki!

[![Description](https://static.igem.wiki/teams/5588/pagelogo/description.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)

[![Design](https://static.igem.wiki/teams/5588/pagelogo/design.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)
[Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)

[![Team Members](https://static.igem.wiki/teams/5588/pagelogo/team-members.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)
[Team Members](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27members%27%29%20%7D%7D)

[![Attributions](https://static.igem.wiki/teams/5588/pagelogo/attributions.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)
[Attributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

[![Collaborations & Partnership](https://static.igem.wiki/teams/5588/pagelogo/collaborations-partnership.webp)](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaborationsandpartnership%27%29%20%7D%7D)
[Collaborations
& Partnership](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaborationsandpartnership%27%29%20%7D%7D)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

SZU-China

2025
SZU-C
hina
2025

↑

![](https://static.igem.wiki/teams/5588/pagelogo/engineering.webp)

# Engineering

Engineering is the application of science to solve real-world problems.

![](https://static.igem.wiki/teams/5588/pagelogo/engineering.webp)

### Engineering

* [overview](#section1)
* [RNAi Silence module](#section2)
  + [RNAi V1: Single-target dsRNA](#rnai-v1)
  + [RNAi V2: Fusion multi-target dsRNA](#long-term-prevention)
  + [RNAi V3: tri-shRNA](#rapid-treatment)
  + [RNAi V4: bi-amiRNA](#in-vitro-transcription)
* [MS2 VLP module](#section3)
  + [1st-gen VLP](#section3-1)
  + [2nd-gen VLP](#section3-2)
* [Cell-free synthesis module](#section4)
  + [CFS-I](#section4-1)
  + [CFS-Ⅱ](#section4-2)
* [References](#section5)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# overview

The APHiGO project consists of **three major engineered modules**: First, the **early
detection system** employs
engineered *Bacillus subtilis* as a **biosensor**, enabling it to detect **aphid
honeydew** and convert it into
the
volatile signal **methyl salicylate (MeSA)**. This system is coupled with an **intelligent
sensor network** for
**real-time monitoring** and early warning through a **WeChat mini-program**,
achieving early detection and monitoring
of aphid infestation during the latent period. Secondly, the **long-term prevention system**
utilizes a **trunk
injection strategy**, where iteratively optimized **RNAi molecules** target key aphid
genes. These RNAi molecules
are delivered via **MS2 viral-like particles** fused with **penetrating peptides**
and **targeting peptides** to ensure
efficient entry into aphid intestinal cells, significantly enhancing both control efficacy and stability.
Finally, for aphid outbreaks, the **rapid treatment system** uses engineered *Metarhizium*
fungi to deliver
a **"dual
strike"**—combining **fungal infection** with continuous expression of **RNAi
molecules**. A **light-controlled safety
switch** is integrated to ensure environmental containment of the strain and effectively prevent its
spread.
Additionally, the project optimizes an **in vitro transcription system**, ensuring large-scale
production of
**high-purity dsRNA**.

In our project, we follow the **DBTL (Design-Build-Test-Learn) principle**, iteratively
optimizing each module.
This forms the core of our engineered approach. During the design phase, we rely on **biological
knowledge**,
**computational models**, **communication with experts**, and **experimental
results**. Each cycle of building,
testing, and learning directly feeds back into a new, more targeted design, creating a **spiral of
continuous
optimization**. It is through this ongoing **DBTL cycle** that we have successfully
transformed an initial concept
into the highly efficient and safe **APHiGO**.

![](https://static.igem.wiki/teams/5588/engineering/dbtl.webp)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# RNAi Silence module

In the RNAi Silence module, we have designed a total of 4 generations of RNAi molecules. Below, you can read
in detail
about our Engineering process.

![](https://static.igem.wiki/teams/5588/design/design-part2-1.webp)

**Please click the collapsible boxes below to view our detailed DBTL cycle process**

### RNAi V1: Single-target dsRNA

▼

#### **Design**

**RNAi V1**: The brown citrus aphid (*Toxoptera citricida*) poses a severe threat
to the citrus industry.
During
in-depth discussions with our principal investigator, Professor Mo, at the early stage of the project,
we learned that RNA-based pesticides, as an emerging green pest control technology, combine high
efficiency with environmental safety—making them an ideal approach for the precise management of brown
citrus aphids. Consequently, we established the use of RNA interference (RNAi) technology as the core
strategy for pest control.

Through cross-screening across multiple databases and performing sequence conservation analysis of
key genes using the MEGA software, we ultimately identified three genes essential to the growth and
development of ***Toxoptera citricida*: chitin synthase (*CHS*), cytochrome P450
(*CYP450*), and cuticle
protein *CP19* (*CP*).** Considering that double-stranded RNA (dsRNA)
represents the most
mature and widely
applied form of RNAi technology in both research and commercial RNA-based pesticides, we constructed
three single-target dsRNA molecules in the RNAi V1 phase—dsCHS, dsCYP450, and dsCP—each designed to
specifically silence one of the three target genes.

To meet the demand for large-scale dsRNA production during the laboratory phase, we employed a
cell-free production system. Plasmids containing the target sequences were first introduced into
*Escherichia coli* for amplification. The extracted plasmids then served as templates for in
vitro
transcription to synthesize the required dsRNA molecules.

For efficacy evaluation, we adopted a plant-mediated RNAi approach, which most closely simulates the
natural oral ingestion of dsRNA and the subsequent gene silencing process in insects. This method
allows for a scientifically rigorous assessment of the lethality and biological function of our
designed RNAi molecules under conditions approximating real-world application.

#### **Build**

During the Build stage, we successfully transformed the constructed recombinant plasmid into *E.
coli* for amplification.
After large-scale plasmid extraction, linearized DNA fragments were obtained through double digestion,
which served as
the standard template for in vitro transcription. After digestion, we used a bidirectional promoter
cell-free production
system to produce the target dsRNA molecules. Through quantitative analysis of the products using
Nanodrop and
qualitative analysis of the products using agarose gel electrophoresis (Fig 1), we finally obtained
the target
single-target dsRNA molecules.

![Agarose gel electrophoresis verification](https://static.igem.wiki/teams/5588/results/3-9.webp)

Fig 1. Agarose gel electrophoresis verification of dsCHS transcription products.
Lanes 1-6 dsCHS: 300 bp. Marker (left): TAKARA DL500 DNA Marker; Marker (right): TAKARA DL1000 DNA
Marker.

To evaluate the gene-silencing efficiency of the RNAi molecules within aphids, we designed and
synthesized multiple pairs of qPCR primers targeting the three genes of interest (*CHS*,
*CYP450*, and
*CP19*) as well as the internal reference gene EF1α. Using conventional PCR and electrophoretic
analysis
(Fig. 2), we preliminarily assessed the amplification efficiency and product size of each primer pair.
Based on these results, TCiCP3, TCiCHS-9, and TCiCYP-14 were selected as the optimal detection primers
for *CP19*, *CHS*, and *CYP450*, respectively. Correspondingly, TCiEF1α-2,
TCiEF1α-14, and TCiEF1α-5 were
chosen as internal reference primers for subsequent qRT-PCR experiments.

![PCR primer validation results](https://static.igem.wiki/teams/5588/results/3-6.webp)

Fig 2. qPCR primer validation result. (a) shows the agarose gel electrophoresis results of
conventional PCR for the positive
reactions of primers *CHS*1–8 and their negative control (NC); (b) shows the
electrophoresis
results for the positive reactions of primers sets *CHS*9–12 and *CP*1–4, along with
their negative
control (NC); (c) shows the results for the positive reactions of primer sets *CP*5–7
and
*CYP450* 1–5, along with their negative control (NC); and (d) shows the results for
the
positive reactions of primers sets *CYP450* 8–13 and their negative control (NC).

#### **Test**

For the experimental data, we corrected the mortality data according to the Abbott formula. Finally,
verification showed that at 120 hours, the corrected mortality rate of aphids in the 400 ng/μl
ds*CHS* treatment group was 11.22%; while that in the 800 ng/μl ds*CHS* treatment group
was 20.48%. The corrected mortality rate of aphids in the 800 ng/μl ds*CP* treatment group
reached 30%, and that in the ds*CYP450* treatment group further increased to 31.4% (Fig. 3).

To further verify the molecular-level effects of RNAi, we extracted total RNA from aphids and
performed qRT-PCR analysis. The results showed that the 800 ng/μL ds*CHS*, ds*CP*, and
ds*CYP450* treatment groups decreased the expression levels of their corresponding target genes
by 27%, 43%, and 30%, respectively (Fig. 4), confirming the effective silencing ability of the
constructed RNAi molecules in vivo.

![Cumulative mortality rates of aphids](https://static.igem.wiki/teams/5588/results/3-11.webp)

Lethality efficiency curve. (a): Cumulative mortality rates of *Toxoptera citricida* (brown
citrus aphids)
treated
with 400 ng/μl dsCHS and 800 ng/μl dsCHS, compared with the control group, at 6 h, 12 h, 24 h, 36 h,
48 h, 60 h,72h,84h,96h,108h and 120 h post-treatment. (b): Cumulative mortality rates of
*Toxoptera citricida* (brown citrus aphids) treated with 800 ng/μl dsCHS, 800 ng/μl dsCYP450,
and 800
ng/μl dsCP, compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h,72h,84h,96h,108h
and 120 h post-treatment.

![Relative expression levels of genes](https://static.igem.wiki/teams/5588/results/3-13-2.webp)

Effects of dsCHS, dsCYP450, dsCP RNA interference (RNAi) on CHS, CYP450, CP19(CP) expression level.
The relative expression levels of *CHS*, *CYP450*, and
*CP19*(*CP*) in *Toxoptera citricida* after
treatment with 800 ng/μl dsCHS, dsCYP450, and dsCP for 120 h, respectively, compared to the control.
Error bars represent the standard deviation (SD) derived from at least three biological replicates.
All datasets were statistically analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P <
0.001; \*\*\*\*P < 0.0001).

#### **Learn**

Based on the experimental results of the first-generation single-target dsRNA, we found that although
RNAi molecules targeting *CHS*, *CYP450*, and *CP19* could all cause aphid death
and significantly reduce the expression of target genes, there were differences in silencing
efficiency among different targets, and the overall lethality level still did not reach the ideal
control effect.

While designing the first-generation dsRNA, we read literature and communicated extensively. After
discussing with Dr. Chen Ruoyu, we introduced the strategy of fusing multi-target dsRNA. By
simultaneously silencing multiple key genes, a synergistic lethal effect is produced, and the design
of the second-generation molecules was initiated.

In addition, during the cell-free production process, we found that the bidirectional promoter-based
transcription system tended to generate non-specific bands exceeding the expected length, which
affected the purity of the dsRNA products. To address this issue, we optimized the template design and
upgraded the in vitro transcription workflow—details of which are presented in our Cell-Free Synthesis
Module.

### RNAi V2: Fusion multi-target dsRNA

▼

#### **Design**

The experimental results of the first-generation single-target dsRNA molecules (dsCHS, dsCYP450, and
dsCP) confirmed the effectiveness of the RNAi pathway but also revealed its limitations. Specifically,
the insecticidal efficiency of single-gene targeting reached a bottleneck, and literature reports
suggest that prolonged use of single-target dsRNA molecules may lead to the development of pest
tolerance. To overcome these challenges, we introduced a fusion multi-target dsRNA strategy in the
second-generation design.

In the design process, we first developed a statistical algorithm to systematically evaluate the
binding efficiency of different targets and nucleotide sites across multiple RNAi prediction
databases. Guided by both the program's analytical results and literature recommendations regarding
effective interference lengths (~60 bp), we ultimately selected short, high-efficiency dsRNA fragments
of 60–100 bp as core modules. Based on this, we constructed dual-fusion dsRNA (dsF2) molecules
targeting *CHS* and *CYP450*, as well as triple-fusion dsRNA (dsF3) molecules capable of
simultaneously
targeting *CHS*, *CYP450*, and *CP19*, through direct fusion of the optimized
short dsRNA segments.

To assess its biosafety, we aligned the designed fused dsRNA sequence with the transcriptome database
of non-target
organisms to ensure that its off-target risk is controllable.

#### **Build**

During the construction phase, the plasmids pUC57\_dsF2 and pUC57\_dsF3, each containing the target
gene fragments, were transformed into *Escherichia coli* for amplification. The plasmids were
then
extracted and digested with restriction enzymes to obtain linearized DNA templates. Using these
templates for in vitro transcription, we successfully synthesized dsF2 and dsF3 dsRNA products. The
resulting molecules were quantitatively analyzed using a NanoDrop spectrophotometer and qualitatively
verified by agarose gel electrophoresis, confirming that the dsRNA products matched the expected
lengths (Fig. 5).

![Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products](https://static.igem.wiki/teams/5588/results/3-14.webp)

Fig 5. Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products.
(a)dsF2: 402bp, marker(left):sangon 1000bp marker; marker(right):BBI DNA Marker A (25~500 bp);
(b)dsF3: 391bp,
marker:sangon 1000bp marker.

#### **Test**

During the experimental validation phase, we employed a plant-mediated RNAi approach to evaluate the
silencing efficiency and aphid mortality induced by the fusion multi-target dsRNA molecules. RNase
free water treatment was used as the negative control, while the experimental groups
consisted of 800 ng/μL dsF2 and 800 ng/μL dsF3.

The results showed that the dsF2 treatment group achieved a corrected mortality rate of 40.8% at 120
hours, whereas the dsF3 treatment group exhibited a stronger insecticidal effect, with the corrected
mortality rate further increasing to 44.9% (Fig. 6).

At the molecular level, dsF2 treatment reduced the expression of the *CYP450* gene by 31.5%.
In
contrast, dsF3 treatment simultaneously suppressed the expression of *CHS*, *CYP450*,
and *CP19* genes, with
expression levels decreased by 35.3%, 20%, and 45%, respectively (Fig. 6). These findings provide
strong evidence that the fusion multi-target dsRNA molecules can effectively achieve synergistic gene
silencing in vivo.

![Experimental validation results of fusion multi-target dsRNA molecules](https://static.igem.wiki/teams/5588/results/3-16-2.webp)

Fig 6. Lethality efficiency curve & effects of dsCHS, dsCYP450, dsCP RNA interference (RNAi) on CHS,
CYP450,CP19(CP) expression level.
(a): Cumulative mortality rates of brown citrus aphids treated with 800 ng/μl dual-fusion dsRNA
(dsF2) or triple-fusion dsRNA (dsF3) at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h,
and 120 h, compared with the control. (b): Relative expression levels of *CHS* and
*CYP450* in
brown citrus aphids treated with 800 ng/μl dsF2 for 120 h, compared with the control.Error bars
represent the standard deviation (SD) derived from at least three biological replicates. All datasets
were statistically analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P <
0.0001). (c): Relative expression levels of *CHS*, *CYP450*, and
*CP19*(*CP*) in brown citrus aphids
treated with 800 ng/μl dsF3 for 120 h, compared with the control.Error bars represent the standard
deviation (SD) derived from at least three biological replicates. All datasets were statistically
analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001).

#### **Learn**

Based on the results of the second-generation fusion dsRNA experiments, we found that the
multi-target synergistic strategy effectively enhanced insecticidal efficacy. However, through further
discussions with field experts, we recognized that long-chain dsRNA molecules are prone to degradation
by RNases in complex environments and may pose potential off-target risks, both of which limit their
practical applications.

To address these challenges, in our **third-generation design**, we introduced a
**short hairpin RNA
(shRNA) structure** to improve molecular stability.

Meanwhile, following in-depth discussions with nanocarrier specialist **Dr. Xuedong
Liu**, we explored an
additional stabilization approach—**encapsulation of RNA molecules using MS2 virus-like
particles (MS2
VLPs)**—to further enhance delivery efficiency and environmental stability. Details of this
work can be
found in the MS2 VLP Module.

During the experimental process, we also noted that existing dsRNA design tools lack functional
support for constructing complex multi-target fusion sequences. To overcome this limitation, we
upgraded our initially developed statistical algorithm into a comprehensive design platform named
**APHiGEM**, enabling efficient and rational design of complex RNAi molecules (see
Software page for
details).

### RNAi V3: Multi-tandem shRNA(tri-shRNA)

▼

#### **Design**

In the third-generation RNAi design, inspired by the results of the previous two generations, we
developed a **multi-tandem short hairpin RNA (tri-shRNA)** architecture. This design
integrates multiple shRNA
units—each specifically targeting one of the key genes *CHS*, *CYP450*, and
*CP19*—into a single **tri-shRNA
construct** through tandem linkage using the classical hairpin loop sequence
**TTCAAGAGA**.

This structural design effectively minimizes redundant sequences and reduces off-target risks, while
the stem–loop configuration enhances the overall stability of the RNAi molecule. Furthermore,
**RNAfold
secondary structure simulations** confirmed that each shRNA unit within the tandem system
maintains
structural independence, supporting the rationality and feasibility of the tri-shRNA design.

#### **Build**

We first performed structural simulations of the designed tri-shRNA using **RNAfold**,
and the predicted
secondary structure results (Fig. 7) confirmed the rationality of the design. Subsequently, the
constructed plasmids were transformed into *E. coli* for amplification, and the transcribed
products
were synthesized using a **cell-free expression system**. The resulting **tri-shRNA
molecules** were
successfully obtained and verified to match the expected length (Fig. 8).

![Secondary structure prediction of tri-shRNA](https://static.igem.wiki/teams/5588/results/3-17.webp)

Fig 7. Secondary structure
prediction of tri-shRNA

![Agarose gel electrophoresis verification of tri-shRNA](https://static.igem.wiki/teams/5588/results/3-18-2.webp)

Fig 8. Agarose gel electrophoresis
verification of tri-shRNA in vitro transcription products.
Marker ( left ) : DL5000 DNA Marker, 1 : negative control, 2、3、4 : tri-shRNA transcription
product213bp
Marker ( right ) : D2000 DNA Marker.

#### **Test**

During the experimental validation phase, we employed a **plant-mediated RNAi approach**
to assess the
mortality induced by the tri-shRNA construct. RNase free water treatment served as the
negative control, while the experimental group was treated with **800 ng/μL tri-shRNA**.
The results
showed that the tri-shRNA treatment group achieved a **corrected mortality rate of 49.5% at 120
hours**
(Fig. 9).

![Cumulative mortality rates of brown citrus aphids](https://static.igem.wiki/teams/5588/results/3-19.webp)

Fig 9. Lethality efficiency curve
Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96
h, 108 h, and 120
h after treatment with 800 ng/μl tri-shRNA and in the control group.

#### **Learn**

Based on the experimental results of the **tri-shRNA**, we observed an improvement in
both synergistic
silencing efficiency and molecular stability compared with the previous generations. However, through
discussions with the **HUBU-China** team, we realized that different species exhibit
distinct
RNAi-processing mechanisms, and that the conventional hairpin loop structures may have limited
processing efficiency within aphid cells.

To achieve more efficient **gene silencing**, in our **fourth-generation
design**, we
incorporated the
**endogenous microRNA (miRNA) processing pathway of aphids**. Specifically, we
constructed **artificial
microRNA (amiRNA)** based on the native aphid miRNA scaffold, aiming to enhance both the
processing
efficiency and silencing specificity of RNAi molecules within the target species.

### RNAi V4: Tandem amiRNA(bi-amiRNA)

▼

#### **Design**

Building upon the advantages of **high molecular stability** and **low off-target
risk** observed in shRNA
constructs, we introduced an **artificial microRNA (amiRNA)** design in our
fourth-generation RNAi system.
By precisely embedding specific interference sequences into the endogenous aphid pre-miRNA scaffold,
this strategy effectively harnesses the host's natural RNAi processing machinery to achieve efficient
gene silencing.

To further enhance synergistic interference, we constructed a **dual-tandem amiRNA
structure(bi-amiRNA)**
capable of
simultaneously targeting the two key genes, *CHS* and *CYP450*. This design continues
our multi-target
regulatory strategy while enabling more efficient gene silencing within the pest.

#### **Build**

During the Build phase, we first screened the **miRBase** database and selected two
endogenous aphid
pre-miRNAs, **api-mir-71 and api-mir-3017a**, as biomimetic scaffolds (Fig. 10). The
validated
high-efficiency siRNA sequences targeting *CHS* and *CYP450* were then substituted into
the mature miRNA
regions of these scaffolds. The two units were subsequently linked using an **AGGCAT
linker** to construct
a **dual-tandem bi-amiRNA**. Secondary structure analysis using **RNAfold**
confirmed that each amiRNA unit
maintained structural independence within the tandem configuration (Fig. 11).

![Skeletal structures of api-mir-71 and api-mir-3017a](https://static.igem.wiki/teams/5588/results/3-20.webp)

Fig 10. The screened aphid miRNA
scaffolds. (a) api-mir-71 scaffold (b) api-mir-3017a scaffold

![Secondary Structure Prediction of the bi-amiRNA](https://static.igem.wiki/teams/5588/results/img-v3-02qr-0d736604-39b9-4082-8a47-2e21cc60013g.webp)

Fig 11. Secondary Structure
Prediction of the bi-amiRNA

The recombinant plasmids were transformed into
*Escherichia coli* for amplification, and the **bi-amiRNA molecules** were
subsequently synthesized
using an
**in vitro transcription system**. The resulting products exhibited complete structural
integrity and the
expected molecular length. Their purity and integrity were further verified through **agarose
gel
electrophoresis** (Fig. 12).

![Agarose gel electrophoresis analysis of in vitro transcribed tri-shRNA](https://static.igem.wiki/teams/5588/results/bmi.avif)

Fig 12. Agarose gel electrophoresis
analysis of in vitro transcribed tri-shRNA.Marker ( left ) : DL5000 DNA Marker, 1 : negative
control, 2、3、4 : tri-shRNA transcription product——157bp, Marker ( right ) : D2000 DNA Marker

#### **Test**

During the experimental validation phase, we employed
a **plant-mediated RNAi approach** to evaluate the insecticidal efficacy of the
**bi-amiRNA** construct.
RNase free water treatment served as the negative control, while the experimental group
was treated with 800 ng/μL **bi-amiRNA**. The results showed that the
**bi-amiRNA** treatment group achieved a
**corrected mortality rate of 60.2% at 120 hours** (Fig. 13). Among all four generations
of RNAi
molecules, **bi-amiRNA demonstrated the highest insecticidal efficiency**, representing
the most effective
construct in our RNAi series.

![Cumulative mortality rates of brown citrus aphids after bi-amiRNA treatment](https://static.igem.wiki/teams/5588/results/3-23.webp)

Fig 13. Lethality efficiency curve
Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96
h, 108 h, and 120
h after treatment with 800 ng/μL bi-amiRNA and inthe control group.

#### **Learn**

Based on the experimental results of the
fourth-generation bi-amiRNA, we successfully validated the advantage of the biomimetic design in
enhancing RNAi efficiency. Both the **lethal effect** and **response speed**
of bi-amiRNA were significantly
superior to those of the previous three RNAi systems (Fig. 14).

![Cumulative mortality rates of brown citrus aphids treated with different RNAi constructs](https://static.igem.wiki/teams/5588/results/3-25.webp)

Fig 14. Cumulative mortality rates
of *Toxoptera citricida* (brown citrus aphids) treated with 400 ng/μl dsCHS, 800 ng/μl dsCHS,
dsCYP450,
dsCP, dsF2, dsF3, tri-shRNA, and bi-amiRNA, compared with the control group, measured at 6, 12, 24,
36, 48, 60, 72, 84, 96, 108, and 120 hours post-treatment.

However, in field applications, RNAi formulations
still face challenges related to **persistence and durability**. To achieve more
effective pest control
under field conditions, we explored the integration of **biological delivery systems**
with RNAi
technology by employing **engineered *Metarhizium anisopliae*** as a living
carrier. This approach
establishes a **dual insecticidal mechanism** that combines fungal infection with
RNAi-mediated gene
silencing. Such a fusion strategy not only leverages the fungus's ability to **colonize crop
surfaces
persistently**, but also enables **specific silencing of target genes**, thereby
offering a **novel and
sustainable pathway** for the green management of citrus aphids.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# MS2 VLP module

From the RNAi V2 experiments, we recognized that **enhancing the environmental stability of RNAi
molecules** is
crucial for
their practical application. To address this, we introduced **MS2 virus-like particles (MS2
VLPs)** as a
**protective delivery
carrier**. Leveraging their self-assembly properties, MS2 VLPs can efficiently encapsulate RNAi
molecules
containing the
**pac site**, thereby preserving the **structural integrity and functional
activity** of RNAi constructs under complex
environmental conditions.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## 1st-gen VLP

#### **Design**

To enhance the stability of RNAi molecules in complex
environmental conditions, we constructed an **in vivo self-assembling delivery system** based
on **MS2 virus-like
particles (VLPs)**. This system exploits the **specific recognition** between the MS2
coat protein and the pac
site on RNA molecules. By co-expressing the coat protein and pac site–containing RNAi molecules in
*Escherichia coli*, the two components spontaneously assemble intracellularly to form
**structurally
complete
VLP–RNAi complexes**.

This design not only provides **effective protection
against
environmental nuclease degradation** but also features **nanoscale particle size**,
which facilitates efficient
**delivery and uptake within biological systems**. Collectively, this MS2 VLP–based strategy
offers a **robust
protective platform** for advancing the practical application of RNAi technology.

#### **Build**

We successfully constructed the recombinant plasmid **pACYCDuet\_1-CP-HisTag-CP-amiRNA**, which
contains the **MS2
coat protein dimer sequence**, a His-tag, and the **target amiRNA expression
cassette**. A pac site was fused to
one end of the target amiRNA to facilitate specific encapsulation. After sequence verification confirmed the
correct plasmid construction, the recombinant vector was transformed into *Escherichia coli*
**BL21(DE3)** for
expression.

To verify the successful encapsulation of the target RNA by VLPs, we performed a **nuclease
protection assay**.
Following gradient nuclease treatment, the RNA within the encapsulated group remained intact, whereas free
RNA was completely degraded. **Coomassie Brilliant Blue staining** simultaneously confirmed the
stable presence
of coat proteins, demonstrating that the assembled VLP complexes effectively protected the RNA payload.

To further assess the **morphology of the virus-like particles, we conducted transmission electron
microscopy
(TEM)** analysis. The purified samples displayed uniform spherical particles with well-defined
boundaries
under negative staining conditions (Fig. 17A, B), confirming the **successful assembly of complete
amiRNA-loaded MS2 VLPs** (Fig. 17).

![TEM images of MS2 VLPs](https://static.igem.wiki/teams/5588/results/4-9.webp)

Fig 17.
Transmission Electron Microscopy (TEM) images of purified histidine-tagged MS2 phage-like particles
(histidine-tagged VLPs). The scale bar represents 100 nanometers.

#### **Test**

We evaluated the protective performance of the VLPs through a **nuclease resistance assay**.
Naked RNA and
VLP-encapsulated RNA were separately treated with **100 U/mL Benzonase Nuclease** at 37 °C. The
electrophoresis
results showed that naked RNA was completely degraded within 10 minutes, whereas the RNA encapsulated within
VLPs remained **intact even after 30 minutes of treatment** (Fig. 18). These findings
demonstrate that the VLPs
provide an effective nuclease barrier for RNA molecules, significantly enhancing their **stability
under
complex environmental conditions**.

![Nuclease resistance assay](https://static.igem.wiki/teams/5588/results/4-10.webp)

Fig 18.
Agarose gel electrophoresis demonstrating the nuclease resistance of amiRNA encapsulated in virus-like
particles (VLPs). Lanes 1–4: 50 µM VLPs treated with 100 U/mL Benzonase nuclease for 0, 10, 20, and 30
minutes, respectively; Lanes 6–9: 500 nM naked amiRNA treated under the same conditions for the
corresponding durations. Results show that VLPs effectively protect amiRNA from degradation under high
nuclease conditions, whereas naked amiRNA is completely degraded within 10 minutes, confirming the
significant protective effect of VLPs on RNA.

#### **Learn**

The first-generation MS2 VLP system successfully achieved **efficient in vivo self-assembly
encapsulation** of
RNAi molecules, and nuclease protection assays confirmed its ability to effectively maintain RNA stability
under complex environmental conditions. However, **transmission electron microscopy (TEM)** and
**gel mobility
analyses** revealed that the assembled VLP complexes exhibited a relatively large particle size
(approximately
**27 nm**), which may limit their tissue penetration and cellular delivery efficiency in
biological systems.

To overcome this bottleneck, the **next-generation system** will incorporate a functional
peptide modification
strategy: by fusing the TAT cell-penetrating peptide to enhance intracellular delivery capability, and
introducing the **GBP3.1 gut-targeting peptide** to improve VLP accumulation efficiency in the
**aphid midgut
tissues**. This design aims to retain the excellent protective properties of the original VLPs
while further
strengthening their **targeting specificity and cellular internalization efficiency**.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## 2nd-gen VLP

#### **Design**

To enhance the targeted delivery efficiency of VLPs within aphids, we integrated a dual-functional peptide
modification system into the MS2 capsid protein. Specifically, the fusion of the **GBP3.1
gut-targeting
peptide** enables specific accumulation in the aphid midgut, while incorporation of the **TAT
cell-penetrating
peptide** enhances intracellular delivery capacity. Through precise molecular docking and spatial
conformation
optimization, this design preserves the self-assembly capability of the capsid protein and establishes a
second-generation VLP delivery system with **both tissue-targeting and transmembrane transport
functions**.

#### **Build**

During the construction phase, we successfully generated three functional validation vectors:
**pET28a\_GBP3.1-eGFP-TAT-HisTag** (for evaluating the targeting and penetration effects of the
dual-functional
peptides), **pET28a\_eGFP** (as a control vector), and
**pET28a\_CP-GBP3.1-CP-TAT-HisTag** (for expressing the
functionalized capsid protein). These plasmids were individually transformed into *Escherichia coli*
BL21(DE3), and induction expression yielded highly purified target proteins, providing essential materials
for subsequent functional verification experiments.

#### **Test**

To evaluate the targeting and cell-penetrating capabilities of GBP3.1 and TAT peptides within aphids, we
conducted a feeding assay comparing the in vivo distribution of the **GBP3.1-EGFP-TAT fusion
protein** with that
of the **EGFP control**. Live fluorescence imaging (Fig. 19) and immunofluorescence analysis of
tissue sections
(Fig. 20) revealed markedly enhanced green fluorescence signals in aphids treated with the fusion protein,
with fluorescence specifically localized in intestinal and other target tissues. These results demonstrate
that GBP3.1 and TAT synergistically mediate efficient translocation of exogenous proteins across the aphid
gut barrier and facilitate intracellular delivery, while GBP3.1 additionally confers distinct
tissue-targeting specificity.

![In vivo fluorescence imaging of aphids](https://static.igem.wiki/teams/5588/proofofconcept/qiepian.webp)

Fig 19.
In vivo fluorescence imaging of aphids.Left: EGFP control group; Right: GBP3.1-EGFP-TAT experimental
group. The experimental group shows significant green fluorescence throughout the body, while the control
group only exhibits weak autofluorescence from chitin (arrows), demonstrating the effective delivery and
retention of EGFP mediated by the dual-functional peptides. Scale bar: 0.5 mm.

![Immunofluorescence imaging of aphid intestinal tissue sections](https://static.igem.wiki/teams/5588/proofofconcept/wps-2.webp)

Fig 20.
**Immunofluorescence imaging** of aphid intestinal tissue sections.Left: **EGFP control
group**; Right:
**GBP3.1-EGFP-TAT experimental group**. Specific **green fluorescence
aggregation** is observed in the **midgut
region** of the experimental group, indicating the **intestinal targeting capability of
GBP3.1** and the **tissue
penetration/diffusion enhancement by TAT**. Blue: **DAPI nuclear staining**. Scale
bar: 50 μm.

#### **Learn**

In the iterative development of the engineered VLP system, the incorporation of a dual-functional peptide
modification strategy significantly enhanced delivery efficiency within aphids through the synergistic
action of targeting and membrane-penetrating mechanisms. However, the introduction of functional peptides
may partially compromise the self-assembly efficiency of the capsid protein, leading to reduced yield and
stability. To address this, future work will focus on systematically evaluating the assembly efficiency and
structural stability of the modified VLPs, while optimizing the linker region between the functional
peptides and the capsid protein to further improve the reproducibility and robustness of the functionalized
VLP system.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Cell-free synthesis module

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## CFS-I

#### **Design**

To meet the demand for large quantities of **high-purity dsRNA** during multiple iterations of
RNAi experiments,
we designed
and established a **scalable production platform** based on cell-free synthesis technology. This
platform employs
a
**bidirectional T7 promoter** system to efficiently generate structurally intact dsRNA molecules
through a
**one-step
transcription** process, providing a stable and reliable source of RNAi materials for subsequent
functional
validation.

#### **Build**

At the Build stage, the constructed **recombinant plasmids** — **pUC57\_CHS**,
**pUC57\_dsF2**, and **pUC57\_dsF3** — were
transformed into
*Escherichia coli* DH5α competent cells using the heat-shock method. Following ampicillin-resistant
colony
screening,
shake-flask amplification, and plasmid extraction, the linearized DNA templates were obtained through
**double-enzyme
digestion**.

#### **Test**

During the in vitro transcription process, the purified linearized DNA templates were incubated with T7 RNA
polymerase, NTPs, and transcription buffer at 37 °C for 4 hours. Using the **bidirectional promoter
system**,
complementary RNA strands were simultaneously transcribed. The reaction products were subsequently treated
with **DNase I** to remove residual DNA templates, yielding **high-purity double-stranded
RNA** (Fig. 21) suitable
for direct use in downstream functional assays.

![Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products](https://static.igem.wiki/teams/5588/results/3-14.webp)

Fig 21.
Agarose gel electrophoresis verification of dsF2 and dsF3 transcription products.
(a)dsF2:402bp,marker(left):sangon 1000bp marker,marker(right):BBI DNA Marker A (25~500 bp)
(b)dsF3:391bp,marker:sangon 1000bp marker

#### **Learn**

During the establishment and optimization of the cell-free transcription system, we successfully achieved
efficient
synthesis of the target dsRNA, providing stable material support for multiple rounds of RNAi iteration
experiments.
However, product analysis revealed a pronounced **read-through phenomenon** in transcription
systems **lacking
effective
terminators**, leading to the generation of **elongated, non-target RNA fragments**.
These byproducts not only
reduced the
yield of functional dsRNA but also introduced potential **off-target risks** due to nonspecific
sequences. Through
systematic literature research, we incorporated a multi-terminator array to construct a hierarchical
termination signal
system, thereby **improving transcription termination efficiency and overall dsRNA yield**.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## CFS-Ⅱ

#### **Design**

To meet the increasing demand for high-yield and high-purity dsRNA production in future RNAi iteration
experiments, we
introduced a **triple-terminator array (T7UUCG–rrnB T1–T7 terminator)** while retaining the
**bidirectional T7
promoter**. The
cooperative action of these multilayered termination signals effectively **reduced transcriptional
read-through**,
thereby
improving both the purity and yield of the target dsRNA products.
To further refine this strategy, we adopted a **modular design** approach by introducing
restriction enzyme sites
between
adjacent terminator units, enabling the construction of gradient plasmids containing single (1T), double (2T),
and
triple (3T) terminator configurations. By comparing the effects of different terminator arrays on
**transcriptional
accuracy** and product yield, we established a practical foundation for the **precise
regulation** of cell-free
transcription systems.

#### **Build**

Based on the pUC57\_CHS construct, we introduced a triple-terminator cassette to generate
**pUC57\_CHS-tri-terminator**.
Following digestion with different restriction enzymes, linearized DNA templates were prepared and verified by
**agarose
gel electrophoresis**. The results confirmed the successful acquisition of linear templates within
the target
band region
(Fig. 22).

![Agarose gel electrophoresis verification of linearized DNA templates](https://static.igem.wiki/teams/5588/results/3-2.webp)

Fig 22.
Agarose gel electrophoresis analysis
(a):MW:marker1,2:TAKARA DL1000 DNA marker; marker3: BBI DNA Marker A (25~500 bp)
1:*CHS*-1T(456bp);2:*CHS*-2T(594bp)(b)MW:marker(left):TAKARA DL1000 DNA marker; marker(right):
BBI DNA Marker A (25~500 bp) 1:*CHS*-NT(346bp);2:*CHS*-3T(716bp)

#### **Test**

Using the same in vitro transcription system for validation, the results showed that compared with the
first-generation
system, the introduction of the **triple-terminator array** significantly reduced non-specific
transcriptional
bands (Fig.
23). Agarose gel electrophoresis revealed markedly **improved product homogeneity**, confirming
that the
multilayer
termination signals effectively suppressed transcriptional read-through.

![Agarose gel electrophoresis analysis of dsRNA purity and transcriptional yield](https://static.igem.wiki/teams/5588/results/3-3.webp)

Fig. 23.
Agarose gel electrophoresis analysis
(a) Agarose gel electrophoresis analysis illustrating the comparison of dsRNA purity; (b) Agarose gel
electrophoresis analysis comparing transcriptional yield.
MW: molecular weight marker; NT-*CHS*: *CHS* dsRNA transcribed from a template lacking
terminators; 1T-*CHS*: *CHS* dsRNA transcribed from a template containing a single T7UUCG
terminator; 2T-*CHS*: *CHS* dsRNA transcribed from a dual-terminator template containing
tandem T7UUCG and rrnB T1 terminators; 3T-*CHS*: *CHS* dsRNA transcribed from a
triple-terminator template containing tandem T7UUCG, rrnB T1, and T7 terminators.

Meanwhile, RNA samples were diluted with nuclease-free water at 1×, 5×, and 10× concentrations and quantified
using a Nanodrop spectrophotometer. The results showed that dsCHS transcribed from the triple-terminator
template exhibited a significantly higher RNA concentration compared with those transcribed from other
templates (Fig. 24).

![RNA concentration analysis](https://static.igem.wiki/teams/5588/results/3-4.webp)

Fig. 24.
Significance analysis of RNA concentrations for transcription products
(a) Significance analysis of RNA concentrations for NT-, 1T-, 2T-, and 3T-template transcription products at
1× dilution; (b) significance analysis at 5× dilution; (c) significance analysis at 10× dilution; (d) linear
regression curves describing the relationship between dilution ratio and RNA concentration. Regression
equations:
NT-*CHS*：YNT=6317\*X-29.37,R2=0.9967；1T-*CHS*：Y1T=9538\*X+164.8,R2=0.9997；2T-*CHS*：
Y2T=9604\*X-100.3,R2=0.9969；3T-*CHS*：Y3T=11006\*X+240.3,R2=0.9971。ns：p>0.05,\*：p
<0.05,\*\*：p <0.01,\*\*\*：p<0.001,\*\*\*\*：p<0.0001

#### **Learn**

Through a systematic evaluation of different terminator arrays in the cell-free transcription system, we
confirmed that
the **triple-terminator array (3T)** confers a significant advantage in improving both the yield
and purity of
dsRNA. Linear
regression analysis further demonstrated that the 3T template maintained the highest **concentration
stability**
across all
dilution gradients (R² = 0.9971), indicating superior transcriptional efficiency and controllability. This
optimization
not only provides a reliable approach for **large-scale production of high-purity dsRNA** but
also establishes a
solid
technical foundation for the future development of more complex RNA molecules.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

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Wang, Z. G., Qin, C. Y., Chen, Y., Yu, X. Y., Chen, R. Y., Niu,
J., & Wang, J. J. (2024). Fusion dsRNA designs incorporating multiple target sequences can enhance the
aphid control capacity of an RNAi‐based strategy. *Pest Management Science*, *80*(6),
2689-2697.

Rajput, I.A., Ahmed, A.M., Khan, K.A. et al. Multitrophic
interactions between coccinellids and their parasitoids in natural and managed plant systems: host plant
and aphid prey species matter. *Int J Trop Insect Sci* *43*, 2011–2020 (2023).

Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA
(shRNA): design, delivery, and assessment of gene knockdown. *Methods Mol Biol*.
2010;*629*:141-58.

Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA
(shRNA): design, delivery, and assessment of gene knockdown. *Methods Mol Biol*.
2010;*629*:141-58.

Jiang, C., Fu, J., Li, F., Xia, K., Li, S., Chang, L., ... &
Zhang, J. (2024). Plastid engineering with an efficient RNAi delivery system based on bacteriophage MS2
virus-like particles enhances plant resistance to cotton bollworm. *Molecular Plant*,
*17*(7), 987-989.

Dang, M., Wu, L. J., Zhang, S. R., Zhu, J. R., Hu, Y. Z., Yang,
C. X., & Zhang, X. Y. (2023). MS2 Virus-like particles as a Versatile peptide presentation platform:
insights into the deterministic abilities for accommodating heterologous peptide lengths. *ACS
Synthetic Biology*, *12*(12), 3704-3715.

Juliana da Rosa, Américo José Carvalho Viana, Fernando Rafael
Alves Ferreira, Alessandra Koltun, Liliane Marcia Mertz-Henning, Silvana Regina Rockenbach Marin, Elibio
Leopoldo Rech, Alexandre Lima Nepomuceno, Optimizing dsRNA engineering strategies and production in E.
coli HT115 (DE3), *Journal of Industrial Microbiology and Biotechnology*, Volume *51*, 2024,
kuae028.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

SZU-China
2025 SZU-C
hina 2025

↑

![](https://static.igem.wiki/teams/5588/pagelogo/experiments.webp)

# Experiments

Experiments is the specific procedures in our Wetlab process

# Protocol

In this module, we will show you in detail all the operating steps involved in our Wetlab process. You can quickly find the experimental procedure information you need through the following five sections, including "Plasmid Construction", "RNA Interference", "Delivery Systems", "Metarhizium", and "Detection Module". Additionally, you can use the brief introductions or subheadings in each section to help you locate the corresponding operating steps.

## Plasmid Construction

This section covers:
 the methods for constructing target plasmids using conventional molecular biology methods.

Click on this box to learn more

## RNA Interference

This section covers:
 the methods for producing dsRNA or shRNA via in vitro transcription.

Click on this box to learn more

## Delivery Systems

This section covers:
 Construction and characterization of MS2 VLP plasmids and Assay for the protective performance of MS2 VLP

Click on this box to learn more

## Metarhizium

This section covers:
 Preparation of Metarhizium spore suspension and Efficacy verification of Metarhizium recombinant strains

Click on this box to learn more

## Detection Module

This section covers
 Induction of Bacillus subtilis using sucrose and Quantification of methyl salicylate by LC-MS method

Click on this box to learn more

[Overview](#plasmid-overview)
[Transformation Method for ***Escherichia coli***](#plasmid-design)
[Method for Transformation of Metarhizium](#plasmid-verification)

## Plasmid Construction

In this module, we constructed a total of **12 types of plasmids**. You may refer to the specific experimental methods for the design and transformation of these plasmids. The corresponding function of each plasmid can be found in table 1, while their sequences can be retrieved from the Sequence module of the Experiment webpage. For more detailed information, you may also access the Results page, which allows you to gain a deeper understanding of the design rationale and design outcomes of each plasmid.

![Figure 7](https://static.igem.wiki/teams/5588/experiments/plasmid.avif)

## Transformation Method for ***Escherichia coli***

### 1. Plasmid Transformation

1. Take the competent cells out of the **-80°C** refrigerator, thaw them on ice, and then transfer them to a biosafety cabinet.
2. Thaw the centrifuge tube containing **4 μg** of plasmid on ice, then place it in a centrifuge and centrifuge at **5000 rpm for 1 min**. After centrifugation, add **40 μL** of sterile water to the tube, mix thoroughly, and adjust the plasmid concentration to **100 ng/μL**.
3. Add **10 μL** of the 100 ng/μL target plasmid solution to **100 μL** of competent cells, mix by gently flicking the bottom of the tube, and incubate on ice for **25 min**.
4. Place the centrifuge tube in a **42°C** metal bath for heat shock treatment (**45 seconds**), then immediately transfer it to ice and incubate for **3 minutes**.
5. Add **700 μL** of antibiotic-free LB liquid medium (prepared as shown in Table 1) to the transformed competent cells, mix well, and place the mixture in a **37°C shaker (220 rpm) for 60 minutes** to recover the bacterial cells.

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-02-02.webp)

### 2. Sectional Streaking and Colony Verification

1. Transfer the resuscitated competent cell suspension to a centrifuge and centrifuge at **5000 rpm for 1 min**. Remove part of the supernatant, leaving approximately **100 μL** of supernatant to resuspend the bacterial pellet. After suspending the bacteria, dip a small amount of the bacterial suspension with a sterile inoculating loop and perform sectional streaking on the surface of LB agar medium containing antibiotics (formulated as specified in Table 3). After streaking, invert the plate and incubate overnight at **37°C** in an incubator.
2. Add an appropriate amount of antibiotics to **5 mL** of antibiotic-free LB broth to prepare antibiotic-containing LB broth. Subsequently, pick a single colony into the aforementioned medium and incubate in a **37°C orbital shaker at 220 rpm** until the OD value reaches **0.8**.
3. A small amount of the bacterial culture is used for PCR reactions (the specific reaction system is shown in Table 4) to confirm that the target plasmid has been successfully transformed into ***Escherichia coli***. The primers used were purchased from **Sangon Biotech Co., Ltd.**, and **2×Taq Master Mix** was purchased from **Vazyme Biotech Co., Ltd.**

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-03-03.webp)

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-04-04.webp)

## Method for Transformation of Metarhizium

### Amplification of Target Plasmids

1. Retrieve DH5α competent cells from **-80°C**, quickly place them on ice, and let stand for **5 min** to allow the cell pellet to thaw.
2. In a laminar flow hood, pipette **5 μL (10~100 ng)** of the target plasmid, add it to the competent cells, gently tap the bottom of the EP tube with fingers to mix, and incubate on ice for **25 min**.
3. Perform heat shock in a **42°C water bath for 45 seconds**, then immediately return to ice and let stand for **2 min**.
4. Add **700 μL** of sterile antibiotic-free LB broth to the centrifuge tube, mix well, and place in a **37°C shaker**; incubate with shaking at **200 rpm for 1 h**.
5. Centrifuge at **5000 rpm for 1 min** to harvest the bacterial cells, discard approximately **600 μL** of the supernatant, gently resuspend the cell pellet by pipetting with the remaining LB broth, and spread onto LB agar medium containing kanamycin (knaR) at a working concentration of **50 μg/mL**.
6. Incubate in a **37°C incubator for 24 h**. Pick a single colony into **500 μL** of sterile water, and heat in a **100°C heat block for 10 min**.
7. Perform PCR identification according to the system (Table 3) in the instructions.
8. Take the bacterial culture and **50% glycerol**, mix them thoroughly at a **1:1 ratio**, and store in a **-80°C freezer**.

### Transformation of Vector into *Agrobacterium tumefaciens* AGL1

1. Retrieve *Agrobacterium tumefaciens* AGL1 competent cells from **-80°C**, quickly place them on ice, and let stand to allow the cell pellet to thaw.
2. In a laminar flow hood, pipette **5 μL** of the vector plasmid obtained in the previous step into **50 μL** of competent cells, gently tap the tube wall with fingers to mix, and incubate on ice for **5 min**.
3. Transfer the mixture to liquid nitrogen for **5 min**, then incubate in a **37°C water bath for 5 min**, immediately return to ice, and let stand for **5 min**.
4. Add **700 μL** of sterile antibiotic-free LB broth, mix well, and place in a **28°C shaker**; incubate with shaking at **200 rpm for 2–3 h**.
5. Centrifuge at **6000 rpm for 1 min** to harvest the bacterial cells, discard approximately **600 μL** of the supernatant, gently resuspend the cell pellet by pipetting with the remaining LB broth, and spread onto LB agar medium containing kanamycin sulfate (working concentration: **50 μg/mL**).
6. After labeling, invert the plate and incubate in a **28°C incubator for 48 h**.
7. Once single colonies grow on the plate, pick a single colony and inoculate it into fresh LB broth containing kanamycin sulfate (working concentration: **50 μg/mL**). After incubating with shaking overnight, take **3 mL** of the bacterial culture for PCR verification; the system and protocol refer to Step 7 in Section 6.3.
8. Take the bacterial culture and **50% glycerol**, mix them thoroughly at a **1:1 ratio**, and store in a **-80°C freezer**.

### Transformation and Screening of *Metarhizium*

#### Transformation of *Metarhizium* by Agrobacterium-mediated transformation method

1. After verification of *Agrobacterium*, take an appropriate amount of the bacterial culture and add it to LB broth containing kanamycin (working concentration: **50 μg/mL**). Incubate in a **28°C shaker at 200 rpm for 12 h**.
2. Collect **6 mL** of the bacterial culture and centrifuge at **4000 rpm for 10 min at 4°C**. Discard the supernatant, resuspend the cell pellet with **1 mL** of IM liquid medium, transfer to a 50 mL centrifuge tube, then add **20 mL** of IM liquid medium (prepared as shown in Table 5-2). Adjust the concentration to an OD660 of approximately **0.2**.
3. Wrap the centrifuge tube with paper, place it in a shaker at **28°C**, and incubate with **200 rpm for 6 hours** until the OD600 reaches approximately **0.6**.
4. Take **200 μL** of the aforementioned bacterial suspension and mix it thoroughly with **200 μL** of spore suspension (concentration: **1×106 spores/mL**).
5. Take **50 μL** of the mixed solution and spread it evenly on IM solid medium (prepared as shown in Table 5-5) with a sterile filter membrane placed on top. Incubate in a **28°C incubator in the dark for 48 hours**.

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-05-05-01.webp)

6. Remove the filter membrane from the IM solid medium and place it on m-100 solid medium (prepared as shown in Table 5-9).

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-05-05-02.webp)

7. The spore suspension is prepared and then spread separately onto PDA and SDAY (prepared as shown in Table 5-6) media containing glufosinate-ammonium and cephalosporin for further cultivation.

### Verification of Recombinant *Metarhizium* Strains

#### Genomic DNA Extraction of Recombinant *Metarhizium* Strains

1. When the mycelia grow to half the area of the PDA Petri dish, scrape the mycelia into **1.5 mL** microcentrifuge tubes using pipette tips.
2. Evaporate the moisture from the microcentrifuge tubes in a **60°C heat block**. After quick-freezing with liquid nitrogen, grind the mycelia into a powder using a grinding rod.
3. Add **400 μL** of Buffer Digestion and **4 μL** of β-mercaptoethanol, then vortex to mix thoroughly.
4. Transfer the tubes to a **65°C water bath** and incubate for **1 h** until the cells are completely lysed. During the water bath incubation, invert the tubes to mix once every **10 min**.
5. After the water bath, add **200 μL** of Buffer PF, invert thoroughly to mix, and then place the tubes in a **-20°C freezer for 5 min**.
6. After the cold treatment, centrifuge at **10,000 rpm for 5 min** at room temperature. Transfer the clear supernatant to a new 1.5 mL microcentrifuge tube.
7. Add **600 μL** of isopropanol to the supernatant, invert approximately **8 times** to mix well, and let stand at room temperature for **3 min**. Subsequently, centrifuge at **10,000 rpm for 5 min** at room temperature and discard the supernatant.
8. Add **1 mL of 75% ethanol** to the pellet, invert to rinse for **2 min**, centrifuge at **10,000 rpm for 2 min**, and discard the supernatant.
9. Repeat Step 8 once.
10. Open the tube lid and invert the tube at room temperature for **10 min** until the residual ethanol is completely evaporated.
11. Dissolve the obtained DNA in **60 μL** of TE Buffer. The extracted DNA is stored at **-20°C** and can be used for subsequent PCR identification and restriction enzyme digestion identification.

#### Genomic PCR Verification

Perform PCR identification in accordance with the following system and protocol (Table 6).

![Figure 7](https://static.igem.wiki/teams/5588/experiments/fig-06-06.webp)

[Overview](#rnai-overview)
[Plasmid Extraction](#rnai-design)
[dsRNA Synthesis](#rnai-validation)
[shRNA Synthesis](#shrna-synthesis)

## RNA Interference Verification

This section outlines the complete workflow for RNA interference verification, including plasmid extraction, dsRNA and shRNA synthesis, and functional validation through aphid gene silencing experiments. The protocols detail the preparation of linear templates, transcription reactions, and quantitative analysis of gene silencing efficiency.

## Plasmid Extraction

Extract plasmids using the **TIANGEN Plasmid Mini Extraction Kit**.

1. Add **500 μL** of Equilibration Buffer BL to Adsorption Column CP3 (place the adsorption column into a collection tube), centrifuge at **12,000 rpm for 1 min**, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.
2. Take **1–5 mL** of overnight-cultured bacterial culture, add it to a centrifuge tube, centrifuge at **12,000 rpm for 1 min** using a conventional benchtop centrifuge, and aspirate the supernatant as much as possible.
3. Add **250 μL** of Solution P1 (RNaseA should be added first) to the centrifuge tube with the bacterial pellet remaining, and thoroughly resuspend the bacterial pellet using a pipette or vortex mixer.
4. Add **250 μL** of Solution P2 to the centrifuge tube, gently invert it up and down **6–8 times** to fully lyse the bacteria.
5. Add **350 μL** of Solution P3 to the centrifuge tube, immediately invert gently up and down **6–8 times** to mix thoroughly; white flocculent precipitates will appear at this time. Then centrifuge at **12,000 rpm for 10 min**.
6. Transfer the supernatant collected in the previous step to Adsorption Column CP3 (place the adsorption column into a collection tube) using a pipette, taking care to avoid aspirating the precipitate. Centrifuge at **12,000 rpm for 30–60 sec**, discard the waste liquid in the collection tube, and place Adsorption Column CP3 back into the collection tube.
7. Add **600 μL** of Wash Buffer PW (absolute ethanol should be added first) to Adsorption Column CP3, centrifuge at **12,000 rpm for 30–60 sec**, discard the waste liquid in the collection tube, and place Adsorption Column CP3 back into the collection tube.
8. Repeat the previous step.
9. Place Adsorption Column CP3 into the collection tube, centrifuge at **12,000 rpm for 2 min** to remove residual wash buffer in the adsorption column. Open the lid of Adsorption Column CP3 and place it at room temperature for **20 min** to completely air-dry residual wash buffer in the adsorption material.
10. Place Adsorption Column CP3 into a clean centrifuge tube, add **60 μL** of RNase-free water to the middle of the adsorption membrane, let stand at room temperature for **2 min**, then centrifuge at **12,000 rpm for 2 min** to collect the plasmid solution into the centrifuge tube.
11. Use **1 μL** of the sample for Nanodrop analysis to preliminarily determine the presence of nucleic acids in the eluate, with RNase-free water as the blank control.
12. Prepare the double enzyme digestion system according to Table 1 below, gently mix each component with a pipette, collect the mixture by brief centrifugation in a palm centrifuge, and incubate at **37°C for 30 min**.
13. Prepare a **2% (w/v)** agarose gel: weigh **0.6 g** of agarose, add **30 mL** of 1× TAE, heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately **60°C**, add SuperRed dye to a final concentration of **0.01% (v/v)** (**3 μL**). Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
14. Prepare the loading sample: mix **10 μL** of the incubated sample with **2 μL** of 6× Loading Buffer (purchased from Bioshop), and place the mixture on ice.
15. Electrophoresis: Remove the comb from the solidified agarose gel, place the gel into the electrophoresis tank with the loading wells facing the negative electrode, and add TAE electrophoresis buffer. Load **10 μL** of 1000 bp DNA marker and **10 μL** of 10000 bp marker into the loading wells, respectively, and load **12 μL** of each sample. Run at **150 V for 25 min**.

## dsRNA Synthesis

### Preparation of Linear Templates

1. Prepare the single/double restriction enzyme digestion system (as shown in Table Table 7-1) (depending on the restriction enzyme sites set for different templates). Gently mix each component with a pipette, collect by brief centrifugation, and incubate at **37°C for 30 min**. All restriction enzyme sites of the templates used are set as shown in Table 7-2.

![Restriction enzyme digestion system table](https://static.igem.wiki/teams/5588/experiments/fig-07-07.webp)

2. Incubate the prepared reaction system at **37°C for 2–4 hours**. After the reaction, incubate at **72°C for 10 min**, then cool at room temperature for **20 min** for annealing.
3. After the reaction, white precipitates may appear; these are magnesium pyrophosphate formed by free pyrophosphate and magnesium ions during the reaction, which will not affect subsequent experiments. You can choose to add some EDTA to remove it, or centrifuge at **2500×g for 1 min** using a benchtop centrifuge to recover the supernatant.
4. Add **3 μL** of DNase I (**1 U/μL**) and **1 μL** of RNase T1 to the reaction system, incubate at **37°C for 15 min** to digest the linear templates used for in vitro transcription and partial single strands of the RNA product.
5. Prepare a **2% (w/v)** RNase-free agarose gel (selected according to the band length): Weigh **0.6 g** of agarose, add **30 mL** of 1× RNase-free TAE, heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately **60°C**, add **3 μL** of SuperRed dye. Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
6. Prepare the loading sample: Mix **1 μL** of sample, **9 μL** of RNase-free water, and **2 μL** of 6× Loading Buffer, then place the mixture on ice.
7. Electrophoresis: Remove the comb from the solidified agarose gel, then place the gel into the electrophoresis tank. An ice-water bath is used outside the electrophoresis tank to reduce heat generation during gel running. Orient the loading wells toward the negative electrode, and add 1× RNase-free TAE electrophoresis buffer until the gel is submerged by approximately **1 mm**. Load **10 μL** of 500 bp DNA marker into the loading well, and load **12 μL** of each sample. Run at **180 V for 25 min**.
8. Dilute each RNA sample with RNase-free water to an appropriate dilution factor, then measure the concentration using a Nanodrop, with RNase-free water as the blank control.

## shRNA Synthesis

### Preparation of Linear Templates

1. Prepare a **50 μL** restriction enzyme digestion system according to the following system (Table 8).

![Restriction enzyme digestion system for shRNA](https://static.igem.wiki/teams/5588/experiments/fig-08-08.webp)

2. Incubate the system in a **42°C heat block for 30 minutes**.
3. Perform gel electrophoresis using a **2% 1×TAE gel** to verify the correctness of the linear template. Proceed to the next step if the length is correct.

### Transcription Reaction

1. Use the **BELONGBIO T7 Transcription Kit Plus** to transcribe the linear template.
2. As shown in the table below (Table 9), prepare a **20 μL** transcription system in a 1.5 mL microcentrifuge tube:

![Transcription system composition](https://static.igem.wiki/teams/5588/experiments/fig-09-09.webp)

3. Incubate the prepared transcription system in a heat block at **37°C for 4 hours**. After 4 hours, adjust the heat block to **72°C** and incubate for **10 minutes**, then remove the tube.
4. Once the microcentrifuge tube has cooled to room temperature, add **3 μL** of DNaseⅠ to each system, and continue incubating at **37°C for 15 minutes**. Store the resulting product at **-80°C**.
5. Prepare a **2% (w/v)** RNase-free agarose gel (selected according to the band length): Weigh **0.6 g** of agarose, add **30 mL** of 1× RNase-free TAE, and heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately **60°C**, add **3 μL** of SuperRed dye. Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
6. Prepare the loading sample: Mix **1 μL** of sample, **9 μL** of RNase-free water, and **2 μL** of 6× loading buffer, then place the mixture on ice.
7. Electrophoresis: Remove the comb from the solidified agarose gel, then place the gel into the electrophoresis tank. Use an ice-water bath outside the electrophoresis tank to reduce heat buildup during gel running. Orient the loading wells toward the negative electrode, and add 1× RNase-free TAE electrophoresis buffer until the gel is submerged by approximately **1 mm**. Load **10 μL** of 500 bp DNA marker into the loading well, and load **12 μL** of each sample. Run at **180 V for 25 minutes**.
8. Dilute each RNA sample with RNase-free water to an appropriate dilution factor, then measure the concentration using a Nanodrop, with RNase-free water as the blank control.

## RNA Interference of Aphid Genes

### Verification of Aphid Gene Silencing by RNA-Containing Diet

#### Preparation of Stock Solutions for Each Component

1. **Amino acid stock solution (10×):** Weigh 20 g of arginine, 10 g of threonine, 10 g of leucine, 6 g of tryptophan, 30 g of aspartic acid, 5 g of glycine, 1 g of tyrosine, 1 g of γ-aminobutyric acid (GABA), 10 g of histidine, 10 g of lysine, 6 g of phenylalanine, 5.5 g of asparagine, 30 g of glutamine, 5 g of serine, 2.5 g of cysteine, 6 g of methionine, 10 g of valine, 7.5 g of isoleucine, 10 g of glutamic acid, 5 g of alanine, 5 g of proline, and 0.5 g of cystine into a **1 L** conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
2. **Vitamin stock solution (5×):** Weigh 2.5 g of ascorbic acid, 0.125 g of pyridoxine hydrochloride, 0.25 g of calcium pantothenate, 2.5 g of choline chloride, 0.125 g of riboflavin, 0.5 g of nicotinic acid, 0.01 g of biotin, 0.5 g of p-aminobenzoic acid, 0.125 g of thiamine hydrochloride, 0.025 g of folic acid, and 2.5 g of inositol into a **1 L** conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
3. **Salt and trace element stock solution (2×):** Weigh 10 g of potassium dihydrogen phosphate, 0.014 g of calcium dihydrogen phosphate, 0.004 g of ferric citrate, 0.014 g of magnesium sulfate, 0.006 g of copper chloride, 0.026 g of zinc chloride, 4 g of magnesium chloride, 0.034 g of calcium lactate, 0.01 g of sodium dihydrogen phosphate, 0.006 g of sodium chloride, and 0.01 g of manganese chloride into a **1 L** conical flask. Add HPLC-grade ultrapure water to the 1 L mark. Stir to dissolve thoroughly, then aliquot into reagent bottles for later use.
4. **Sugar stock solution (10×):** Weigh **300 g** of sucrose into a 100 mL conical flask. Add HPLC-grade ultrapure water to dissolve, and while stirring, add water to reach 100 mL. Once the sucrose is completely dissolved with no particles, aliquot into reagent bottles for later use.

#### Operation for Nutrient Solution Mixing (100 mL)

Take a **100 mL** conical flask, and sequentially add **10 mL** of amino acid stock solution (10×), **20 mL** of vitamin stock solution (5×), **50 mL** of salt and trace element stock solution (2×), and **10 mL** of sugar stock solution (10×). After mixing, add HPLC-grade ultrapure water to the 100 mL mark.

#### Preparation and Addition of Citrus Juice

Take **5 fresh tender citrus leaves**, sample each leaf using a **1 cm diameter** punch. Mix the citrus leaf samples, add **5 mL** of ultrapure water, and grind in a sterilized mortar. Centrifuge and collect the supernatant for later use. Add **4 mL** of this supernatant to the prepared 100 mL nutrient solution.

#### pH Adjustment and Sterilization

Immerse the pH meter probe into the solution to monitor the pH value (measure after stirring uniformly). Use a pipette to add **1% NaOH or HCl** solution dropwise to adjust the pH to **7.4–7.7**.

Sterilize using a **0.22 μm** microporous filter membrane and matching filtration device. Aliquot filtrate into sterile reagent bottles and store at **-20°C** for later use.

![Nutrient solution mixing table](https://static.igem.wiki/teams/5588/experiments/fig-10-10.webp)

#### Diet Rearing

The aphid rearing method refers to the protocol described by Li et al. (1997), with specific operations as follows: The aphid rearing chambers use open-ended polymethyl methacrylate (PMMA) tubes with a diameter of **3.2 cm** and a height of **3 cm**. Cut Parafilm into **1.5 cm²** pieces, stretch each piece by hand to cover one end of a glass tube, and irradiate it under ultraviolet (UV) light for **15 minutes**. After that, add **150 μL** of artificial nutrient solution to the surface of the Parafilm, and then cover it with two additional layers of Parafilm. Place the aphids into the tube, and repeat the same operation for the other end of the tube. Rear the aphids in an illuminated incubator under the following conditions: temperature of **24°C ± 1°C**, relative humidity of **85%–90%**, and photoperiod of **16:8 (L:D, Light:Dark)**.

The aphid source is collected from field crops; aphids of the same instar or from the same brood of viviparous aphids are selected as the test population. After being starved for approximately **3 hours**, the aphids are transferred into the glass tubes, with **200 aphids per tube**.

### Plant-Mediated RNA Interference

1. Take the young instar aphids required for the experiment, **30 aphids per group**, and starve them for **3 hours** for later use.
2. Cut tender citrus stems of approximately **10 cm** in length, soak them in **75% ethanol** for sterilization, and set aside.
3. Prepare the required RNA concentration using RNase-free water (the concentration can be calibrated by Nanodrop), and take an equal volume of RNase-free water as the control for later use.
4. Prepare the rearing devices needed for the experiment: Take an appropriate number of **1.5 mL and 50 mL** microcentrifuge tubes. Make a neat cut along the bottom edge of the 1.5 mL microcentrifuge tube, ensuring the cut is as even as possible during operation.
5. After labeling the group numbers, add **300 μL** of RNase-free water to the control group. Insert the 10 cm citrus tender stem into the bottom-cut 1.5 mL microcentrifuge tube, and tightly wrap the tube mouth with Parafilm to prevent aphids from accidentally falling in. For the experimental group, replace RNase-free water with the prepared RNA, repeat the above operations until all groups are set up. Place them in a laminar flow hood and let stand for **3 hours** to allow the plants to absorb the liquid.
6. Gently transfer the aphids to the 10 cm citrus tender stems using a writing brush, **30 aphids per group**.
7. Unscrew the lid of the 50 mL centrifuge tube, and use non-toxic glue to vertically fix the 1.5 mL microcentrifuge tube (with the open end facing up) containing the inserted citrus tender stem at the center of the lid. After the glue solidifies, slowly fit the 50 mL centrifuge tube wall over the lid and tighten the lid. Wrap the end of the 50 mL centrifuge tube away from the lid with gauze, then transfer it to an artificial climate incubator for cultivation. The cultivation conditions are strictly set as follows: temperature **25±1°C**, relative humidity **70±5%**, photoperiod **16:8 hours (Light:Dark)**.
8. Observe the growth status of aphids and tender stems every **12 hours**, and record the number of dead aphids.
9. **Rearing maintenance:** Replace the rearing devices every **two days**. In the laminar flow hood, collect dead aphids into new 1.5 mL microcentrifuge tubes, flash-freeze them in liquid nitrogen, and store them in a **-80°C** refrigerator for later use. Gently transfer surviving aphids with a writing brush to new microcentrifuge tubes containing freshly sterilized citrus tender stems (new tubes prepared as per the aforementioned steps) to ensure sustained nutrient supply.
10. After **5 days** of observation, collect all remaining aphids into the 1.5 mL microcentrifuge tubes containing dead aphids as mentioned above, clearly label the group information, flash-freeze them in liquid nitrogen, and store them in a **-80°C** refrigerator for later use.

### Calculation of RNA Lethal Efficiency

Aphids are marked as dead if they fall off the plant and lie on their side, show no antenna movement when stimulated with a writing brush, or have a withered body. After recording the mortality data of the control group and experimental group, correct the mortality data according to **Abbott's formula**.

The specific formula is as follows:

![Abbott's formula for mortality correction](https://static.igem.wiki/teams/5588/experiments/fig-1.webp)

Figure.01

## Quantitative Detection of mRNA Silencing Efficiency of Target Genes

### Method Using Aphid Total RNA Extraction Kit

#### Preparations Before RNA Extraction Experiment

1. Wear a mask and gloves;
2. Environmental treatment: Spray RNA Eraser on all surfaces that may be contacted first, then wipe dry with ethanol;
3. Unseal new pipette tips to be used; (For detailed procedures, refer to Invitrogen's **PureLink™ RNA Mini Kit**)

#### RNA Extraction Procedure

1. Take out the experimental aphid samples previously stored in a **-80°C** refrigerator. Grind the aphids with a manual grinding rod, then add **600 μL** of Lysis Buffer and **6 μL** of β-mercaptoethanol. Centrifuge at **12,000×g for 2 min at 25°C** (room temperature). Transfer the supernatant to a new collection tube and discard the insect cuticle precipitate.
2. Add an equal volume (**600 μL**) of **70% ethanol** to the collection tube containing the supernatant lysis buffer. If precipitates are visible, gently disperse them.
3. Transfer up to **700 μL** of the sample into a spin column (with a collection tube) at a time, and centrifuge at **12,000×g for 15 seconds**. Discard the flow-through, then reinsert the spin column back into the same collection tube.
4. Repeat the previous step to transfer the remaining sample into the same collection tube until all the sample is transferred.
5. Add **700 μL** of Wash Buffer Ⅰ to the spin column, and centrifuge at **12,000×g for 15 seconds** at room temperature. Discard the flow-through and the collection tube, then place the spin column into a new collection tube.
6. Add **500 μL** of Wash Buffer Ⅱ to the spin column, and centrifuge at **12,000×g for 15 seconds** at room temperature. Discard the flow-through, and keep the spin column in the same collection tube.
7. Repeat the previous step once.
8. Centrifuge the empty spin column (with the collection tube) at **12,000×g for 1 min** at room temperature.
9. Add **30–50 μL** of Elution Buffer to the spin column.
10. After standing at room temperature for **1 min**, centrifuge at **12,000×g for 2 min** at room temperature.
11. Repeat the previous step **2–3 times** using the collected eluate.
12. Measure the concentration of the extracted RNA using a Nanodrop; quantify the RNA by measuring the absorbance at **260 nm**. Evaluate the purity of all RNA samples by the absorbance ratios of optical density (OD) **260/280** and OD **260/230**.
13. Confirm the integrity of the RNA by **1% agarose gel electrophoresis**.
14. Store the RNA samples at **-80°C** for later use or proceed directly to reverse transcription.

### cDNA Synthesis

Please refer to the kit: **TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGene)**

#### Reaction System:

![cDNA synthesis reaction system](https://static.igem.wiki/teams/5588/experiments/fig-11-11.webp)

#### Reaction Process:

![cDNA synthesis reaction process](https://static.igem.wiki/teams/5588/experiments/fig-12-12.webp)

### qRT-PCR

#### 1. Reference Gene Selection & Primer Design

![Reference genes and primer sequences](https://static.igem.wiki/teams/5588/experiments/fig-13-13.webp)

#### 2. Setup of the qPCR System

**Configure qPCR system:**

![qPCR reaction system](https://static.igem.wiki/teams/5588/experiments/fig-14-14.webp)

**Paving of a 96-well plate:**

![96-well plate layout](https://static.igem.wiki/teams/5588/experiments/fig-15-15.webp)

#### 3. Running the qPCR Program

![qPCR thermal cycling program](https://static.igem.wiki/teams/5588/experiments/fig-16-16.webp)

#### 4. Data Processing

Calculations were performed using **relative quantification (ΔΔCt method)**.

All measured values are referenced to the value of **1.0** obtained from the RNase-free water (control group) treatment. Error bars represent the standard deviation (SD) derived from at least **three biological replicates**. Statistical analysis of all datasets was conducted using a **one-tailed unpaired t-test** (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001).

[Overview](#delivery-overview)
[Characterization and Purification of Proteins](#delivery-Characterization)
[Assembly and Characterization of VLP Nanoparticles](#delivery-Assembly)
[Red Ink Immersion Experiment](#delivery-RedInk)
[Verification of RNA Stability Protected by MS2](#delivery-Verification)
[Functional Verification of Localization Peptide GBP3.1 and Cell-Penetrating Peptide TAT](#delivery-Functional)

## Characterization and Purification of Proteins

### Bacterial Lysis

1. Centrifuge the induced bacterial culture at **7,000 rpm** for **5 min** at **4°C**, and discard the supernatant. Then resuspend the bacterial pellet in 1× Phosphate-buffered Saline (PBS) solution, centrifuge again, and discard the supernatant.
2. Weigh the empty centrifuge tube. Add non-denaturing lysis buffer (300 mM sodium chloride, 50 mM sodium dihydrogen phosphate) at a ratio of **"4 mL per gram of wet bacterial weight"**, and add Protease Inhibitor Cocktail, Universal, 100× to the lysis buffer, then resuspend the bacteria.
3. Add lysozyme to the bacterial resuspension to a final concentration of **1 mg/mL**, mix gently, and incubate the centrifuge tube on ice for **30 min**.
4. Keep the treated bacterial solution on ice and lyse using an ultrasonic cell disruptor (Xinzhi). Set the ultrasonic power to **200–300 W**, with an ultrasonic mode of **"6 seconds on, 9 seconds off"**, and a total sonication time of **35 minutes**. Maintain an ice bath during lysis to prevent target protein denaturation due to increased temperature of the bacterial solution.
5. After sonication, add an equal volume of nuclease-free water to the bacterial solution to dilute the cation concentration in the system. Then add Benzonase Nuclease to a final concentration of **100 U/mL**. Simultaneously, add MgCl₂ to achieve a final concentration of **2 mM Mg²⁺** in the system to activate the benzonase nuclease, then incubate the bacterial solution at **37°C** for **45 min**.
6. Centrifuge the bacterial solution treated with Benzonase Nuclease at **10,000×g** for **20 min** at **4°C**, collect the supernatant containing the target protein, and filter the supernatant through a **0.22 μm** polyethersulfone (PES) syringe filter to remove unbroken bacteria and cell debris. Collect the filtered supernatant.

### Protein Purification

Purify the protein using Beyotime's His-tag Protein Purification Kit (Reduction-Resistant Chelating Type):

1. According to the ratio of **"4 mL bacterial lysate supernatant per 0.5 mL resin"** (equivalent to 1 mL of 50% resin suspension) (1:8), take an appropriate amount of well-mixed 50% BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type), centrifuge at **1,000×g** for **10 seconds** at **4°C**, and discard the resin storage solution. Add 1 column volume (CV) of non-denaturing lysis buffer to the resin, mix gently to equilibrate the resin, centrifuge again at **1,000×g** for **10 seconds** at **4°C** to discard the liquid, and repeat this equilibration step 1–2 times. Finally, discard the equilibration buffer.
2. Fully mix the equilibrated BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type) with the bacterial lysate supernatant, and incubate on a **4°C** orbital shaker (**80 rpm**) with gentle shaking for **60 minutes**.
3. Load the mixture of resin and lysate supernatant into an empty 12 mL affinity chromatography column tube, open the bottom cap of the purification column, and allow the liquid to flow out naturally under gravity. During this process, collect approximately **20 μL** of the flow-through for subsequent analysis of the binding efficiency between the protein and the resin.
4. Perform **8 column washing steps**: Add 1 CV of non-denaturing washing buffer to the column each time. After the liquid flows out completely, collect **20 μL** of the washing solution for subsequent analysis of protein elution efficiency.
5. After column washing, perform **9 elution steps** for the target protein: Add 1 CV of non-denaturing elution buffer to the column each time. After the liquid flows out naturally, collect each eluate into pre-labeled centrifuge tubes for subsequent experiments.
6. Take **80 μL** of the sample, add **20 μL** of 5× SDS-PAGE Sample Loading Buffer, and vortex for **10 seconds**. Boil in a **100°C** heat block for **5 min** (to promote full protein denaturation), then immediately transfer to ice to cool for **3 min** (to terminate the denaturation reaction). After cooling, centrifuge the sample at **12,000×g** for **1 min**, and load the supernatant onto a **12% polyacrylamide** separating gel for SDS-PAGE electrophoresis. After electrophoresis, stain with Coomassie Brilliant Blue R-250 (solution system as shown in Table 17; Table 18) and destain until bands are clear. Based on the electrophoresis results, select the original samples corresponding to lanes with clear target bands and minimal interference from contaminating bands for subsequent experimental analysis.

![Table 17](https://static.igem.wiki/teams/5588/experiments/fig-17-17.webp)

![Table 18](https://static.igem.wiki/teams/5588/experiments/fig-18-18.webp)

## Assembly and Characterization of VLP Nanoparticles

### Acid Hydrolysis

1. At a ratio of **"target protein to glacial acetic acid of 1:2"**, add twice the volume of glacial acetic acid to the concentrated eluate, and incubate on ice for **30 minutes** to denature the capsid, decompose it into protein dimers, and precipitate RNA. Subsequently, centrifuge at **10,000×g** for **20 minutes** at **4°C** to remove the precipitated nucleic acids.
2. Appropriately dilute the supernatant with ultrapure water and transfer it to an Amicon® Ultra centrifugal ultrafiltration tube (Specification: **10 kDa** molecular weight cut-off (MWCO), sample volume 2 mL, regenerated cellulose membrane). Filter with assembly buffer (42 mM Tris, pH 7.5; 84 mM NaCl; 3 mM acetic acid; 1 mM EDTA, pH 8.0) for **5 times** to remove high-concentration glacial acetic acid, and collect the concentrated sample via reverse centrifugation.
3. According to the manufacturer's instructions, quickly determine the content of the target protein in the solution using the Bradford method.

### In Vitro Assembly

1. Clean 1.5 mL microcentrifuge tubes with RNaseEraser (Forgene), and after air-drying, prepare the assembly system according to Table 19. Mix all components thoroughly, then incubate at **21°C** for **10 minutes**.
2. Add **1 μL** of RNase A to the sample to a final concentration of **100 μg/mL**, incubate for digestion for **30 minutes**. Then add **3 μL** of RiboLock RNase Inhibitor (40 U/μL) at a ratio of **"RNase inhibitor to reaction system of 3/100 (v/v)"** to terminate the enzymatic hydrolysis reaction of RNase A.

![Table 19](https://static.igem.wiki/teams/5588/experiments/fig-19-19.webp)

### Reverse Transcription PCR (RT-PCR)

Perform RT-PCR using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix:

1. Pipette **2 μL** of **0.1 μg/μL** Random Primer (N9) and **14 μL** of assembled VLP nanoparticles into the same 0.2 mL flat-cap thin-walled tube. Mix thoroughly, incubate in a **65°C** heat block for **5 min**, then place on ice for **2 min**.
2. Add **20 μL** of 2× TS Reaction Mix, **2 μL** of RT/RI Enzyme Mix, and **2 μL** of gDNA Remover to the above-treated solution. Mix thoroughly, then place in a Biometra TAdvanced instrument. Set the PCR program as: **"incubate at 25°C for 10 min, then at 42°C for 50 min"**.

### Verification by Agarose Gel Electrophoresis

1. Prepare a **1% (w/v)** agarose gel: Weigh **0.6 g** of agarose, add **60 mL** of assembly buffer (Table 19) prepared with RNase-free water, and heat in a microwave oven to completely dissolve the agarose. When the gel cools to approximately **60°C**, add **3 μL** of SuperRed dye to a final concentration of **0.01% (v/v)**. Select an appropriate gel casting tray, insert the comb, pour the dissolved gel into the tray, and let it stand to solidify.
2. After the gel solidifies, place it in an electrophoresis tank filled with assembly buffer (42 mM Tris, pH 7.5; 84 mM NaCl; 3 mM acetic acid; 1 mM EDTA, pH 8.0). Pipette **40 μL** of the assembled sample into a new 1.5 mL microcentrifuge tube, then add **8 μL** of 6× loading buffer. Mix thoroughly and load the entire mixture into a gel well. Load **10 μL** each of DL1000 marker and DL500 marker. Run electrophoresis at **100 V** for **1 hour**.
3. After electrophoresis, use a UVP Benchtop UV Transilluminator (Analytik Jena GmbH + Co. KG) to verify the presence of RNA in the VLP nanoparticles.
4. Stain with Coomassie Brilliant Blue (prepared as shown in Table 17) for **1.5 hours**, then destain with destaining solution (prepared as shown in Table 18). Repeat destaining until the background is clear.

### Verification by Transmission Electron Microscopy (TEM)

1. **Sample adsorption:** In a laminar flow hood, pipette **30 μL** of the sample solution onto the surface of clean Parafilm. Take a 200-mesh carbon-coated copper grid, place it gently on the sample droplet with the front side (support film side) facing down, and incubate at room temperature for **10 min** to ensure the sample is fully attached to the grid surface.
2. **Sample drying:** Gently touch the edge of the copper grid with the edge of clean filter paper to absorb excess sample solution. Then place the copper grid with the front side facing up in a room-temperature, light-protected environment to air-dry naturally (approximately **30–60 min**). Avoid air disturbance during this period to prevent contamination.
3. **Washing and negative staining:** When the grid surface is in a semi-dry state, immediately place it gently on a **30 μL** ultrapure water droplet with the front side facing down. Absorb excess water using filter paper as described above. Then, transfer the grid (front side down) to a **30 μL** droplet of **2% phosphotungstic acid hydrate** solution (pH 6.5–7.0) and stain at room temperature for **1 min**. After staining, absorb excess stain with filter paper, transfer the grid to a new **30 μL** ultrapure water droplet for **30 s** (to remove excess stain), and finally blot dry residual water with filter paper.
4. **TEM observation:** Place the prepared copper grid on the TEM sample stage, observe under an accelerating voltage of **80 kV**. Adjust the magnification according to the morphological characteristics of the sample, capture clear images, and record relevant parameters.

## Red Ink Immersion Experiment

1. Select stems with similar morphology and consistent physiological status, then cut stem segments of equal length with the same number of leaves. Immerse the segments in a **15 mL** EP tube containing **9 mL** of red ink, and conduct time and concentration gradient experiments.
2. After immersion, peel off the bark of the stems to expose the white phloem, and observe the diffusion of red ink.
3. Make a cross-section at the mid-height of the stem segment, take a photo of the cross-section, and use ImageJ to measure the ratio of the red ink area to the total cross-sectional area for subsequent flow rate calculation.

### Injection Experiment of Red Ink and Protein Mixture

* Select citrus seedlings for stem injection. Use an electric drill and a matching drill bit to drill a hole in the citrus seedling; the depth of the hole should not exceed half the diameter of the trunk.
* Inject **1 mL** of the mixed solution of red ink and VLP nanoparticles (protein concentration: **50 μM**) into a 5 mL trunk syringe, then add a driving piston. Next, inject **2–3 mL** of clean water into the syringe to form a hydraulic driving chamber between the water and the original piston. Use the self-weight of the clean water as a stable external pressure to achieve continuous infusion of the mixed solution.
* Insert the modified syringe mentioned above into the pre-drilled guide hole at a **45° downward angle**, and leave the syringe in the trunk until red color appears in the leaves (Figure 02).

![Figure 02](https://static.igem.wiki/teams/5588/experiments/fig-2.webp)

Figure 02

* Cut approximately **1–1.5 g** of tender stems with new leaves at different heights, grind them thoroughly in liquid nitrogen, and collect the powder.
* Add **800 μL** of non-denaturing lysis buffer (300 mM sodium chloride, 50 mM sodium dihydrogen phosphate) and Protease Inhibitor Cocktail, Universal, 100× to the lysis buffer, then vortex to mix thoroughly.
* Centrifuge at **10,000×g** for **10 minutes** at **4°C** to separate the supernatant.
* Take **80 μL** of the sample, add **20 μL** of 5× SDS-PAGE Sample Loading Buffer, and vortex for **10 seconds**. Boil in a **100°C** heat block for **5 minutes** (to promote full protein denaturation), then immediately transfer to ice to cool for **3 minutes** (to terminate the denaturation reaction). After cooling, centrifuge the sample at **12,000×g** for **1 minute**, and load the supernatant onto a **12% polyacrylamide** separating gel for SDS-PAGE separation.
* Take a nitrocellulose (NC) membrane of the same size as the SDS-PAGE gel, directly place it in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3) to equilibrate for **10 minutes**. Meanwhile, immerse 6–8 layers of qualitative filter paper in transfer buffer until fully soaked. After SDS-PAGE, carefully peel off the gel, cut off unnecessary edges (retain the target protein region), and place it in transfer buffer to equilibrate for **5 minutes**.
* Transfer the proteins from the gel to the NC membrane using a GenScript rapid wet transfer apparatus.
* After transfer, transfer the NC membrane to TBST blocking solution containing **5% BSA**, ensuring the membrane is completely submerged. Block overnight with gentle shaking on a room-temperature shaker (**80 rpm**).
* Discard the blocking solution, and quickly rinse the membrane once with TBST (for **1 minute**). Dilute the anti-His tag primary antibody according to the antibody instructions (**1:1000**, diluted in TBST containing 5% BSA), place the membrane in the primary antibody solution, and incubate for **2 hours** on a room-temperature shaker (**80 rpm**).
* Recover the primary antibody, transfer the membrane to TBST washing solution, and wash for **15 minutes** on a room-temperature shaker (**80 rpm**); repeat this washing step three times.
* Dilute the secondary antibody according to the antibody instructions (**1:5000**, diluted in TBST containing 5% BSA), place the membrane in the secondary antibody solution, and incubate for **2 hours** on a room-temperature shaker (**80 rpm**).
* Recover the secondary antibody, transfer the membrane to TBST washing solution, and wash for **15 minutes** on a room-temperature shaker (**80 rpm**); repeat this washing step three times.
* Mix ECL chemiluminescent substrate Solution A and Solution B at a **1:1 ratio**, evenly add the mixture to the surface of the NC membrane (after blotting residual liquid with lens cleaning paper) to cover the target band region, and let stand in the dark for **1–2 minutes**. Place the membrane in a chemiluminescence imager, adjust the exposure time (**10 seconds – 5 minutes**) according to signal intensity, capture images, and record the target band corresponding to the His-tagged protein (compare the molecular weight with the Marker).

## Verification of RNA Stability Protected by MS2

1. Prepare the Benzonase Nuclease digestion reaction system (volume **100 μL**) according to Table 20, and react for **0 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 120 min** respectively.
2. After the reaction time elapses, add **1 μL** of **50 mM EDTA** to inhibit the activity of Benzonase Nuclease. Then verify the RNA in the above reaction products by agarose gel electrophoresis, and visualize protein bands using Coomassie Brilliant Blue staining solution.

![Table 20](https://static.igem.wiki/teams/5588/experiments/fig-19-19.webp)

## Functional Verification of Localization Peptide GBP3.1 and Cell-Penetrating Peptide TAT

### Feeding Assay

1. Resuspend **5 µg** of the peptide-EGFP fusion protein in **200 µL** of aphid nutrient solution.
2. Add **100 µL** of the aforementioned protein-nutrient solution using the double-membrane method.
3. Feed **20 aphids** per membrane for **24 hours**, with three replicates per treatment.

### Observation of Living Aphids

Pick the fed aphids with a writing brush and place them on a glass slide. Drop absolute ethanol vertically onto the aphids for anesthesia, and let stand for **1 minute** to allow excess absolute ethanol to evaporate. Then, observe the prepared slide using a Zeiss Axioplan II fluorescence microscope with a FITC filter.

### Observation of Frozen Sections

* **Antigen retrieval:** Antigen retrieval is not routinely performed for frozen sections; it can be selectively conducted under special circumstances.
* **Circle marking:** Mark a circle on the section using an immunohistochemical pen, then place the section in TBST.
* **Membrane permeabilization:** Permeabilize the membrane with **0.1% Triton® X-100** for **15 minutes**. Wash 3 times with TBST, **5 minutes** each time.
* **Blocking:** Add **10% goat serum** dropwise, and incubate at **37°C** for **30 minutes**.
* **Primary antibody incubation:** Flick off the serum, dilute the EGFP primary antibody stock solution with TBST to prepare a primary antibody working solution at a **1:500 dilution**. Mix thoroughly using a vortex mixer, then add **50–100 μL** of the primary antibody working solution to each section (depending on tissue size). Incubate at **4°C** over two nights.
* **Secondary antibody incubation:** On the third day, take the sections out of the refrigerator and let them stand at room temperature for **15 minutes** to rewarm. Rinse 3 times with TBST, **3 minutes** each time. Dilute the secondary antibody stock solution with TBST to prepare a secondary antibody working solution at a **1:400 dilution**. Add **50–100 μL** of the secondary antibody working solution to each section (depending on tissue size), and incubate at **37°C** for **45 minutes**. Rinse 3 times with TBST, **3 minutes** each time.
* **Nuclear staining:** Remove TBST, add **50–100 μL** of DAPI working solution (prepared by diluting DAPI stock solution at **1:500**) to each section (depending on tissue size). Stain nuclei in the dark for **5 minutes**, then rinse with TBST.
* **Mounting:** Mount the sections with fluorescent mounting medium, and store at **4°C** in the dark.
* **Microscopic examination:** Examine the sections under a microscope, and perform image acquisition and analysis.

[Overview](#metarhizium-overview)
[Culture of Metarhizium](#metarhizium-culture)
[Efficacy Verification of Recombinant Metarhizium Strains](#metarhizium-assay)

## Culture of Metarhizium

### Colony Morphology

Metarhizium is a small filamentous fungus. On comprehensive Potato Dextrose Agar (PDA) medium, its colonies are distinct: initially white with dense and vigorous hyphae, later producing pale yellow-green spores that spread toward the edge of the petri dish. The reverse side of the medium appears yellow.

### Activation and Cultivation Methods

1. **Medium Preparation:** Use Potato Dextrose Agar (PDA) medium (purchased from Beina Biology) for cultivation. Add **47.0 g** of PDA to **1000 mL** of pure water, autoclave at **121°C** for **20 minutes**, then pour into petri dishes. (Note: Medium components are as follows: potato extract powder: 10.0 g, glucose: 20.0 g, KH₂PO₄: 3.0 g, MgSO₄·7H₂O: 1.5 g, thiamine: 0.008 g, agar: 15.0 g, pH: 6.0±0.2)
2. Take the test tube containing lyophilized powder from **-80°C**, place it on ice in a laminar flow hood. Wipe the outer tube with cotton soaked in **75% ethanol**, heat the top with an alcohol lamp, then quickly drop sterile water to crack the top. Subsequently, use tweezers to break it open, and remove the inner tube and cotton plug.
3. Aspirate **0.5 mL** of sterile water into the lyophilization tube to fully dissolve the fungal powder.
4. Transfer the solution to a microcentrifuge tube (EP tube), repeat the process with another **0.5 mL** of sterile water, seal the tube, and let it stand at room temperature for **1 hour**.
5. Add **200 μL** of the fungal solution to each petri dish and spread it evenly.
6. Incubate the petri dishes at **25–28°C** for **3–5 days**; the strain can be used once it grows.

### Preparation of Metarhizium Spore Suspension

Metarhizium can produce sufficient spores after culturing on PDA medium for approximately **1 week**. Further prepare the spore suspension for subsequent experiments such as transformation, following these steps:

1. Prepare **0.05% Tween-80**, place it in an autoclave, and sterilize at **121°C** for **20 minutes**.
2. Take a petri dish with well-grown Metarhizium, add **6 mL** of sterilized 0.05% Tween-80 to the dish, gently scrape the surface of the colony with a pipette tip or spreader to detach the spores, and prepare a crude spore suspension.
3. Take a double-ended plastic tube, cover one end with 2 layers of gauze. Aspirate the crude spore suspension with a pipette, press the pipette tip against the gauze to expel the liquid. Remove hyphae through 2 layers of sterilized gauze to obtain the spore suspension.
4. Determine the spore concentration using a hemocytometer.
5. Add an appropriate amount of 0.05% Tween-80 according to the concentration, dilute to the required concentration, and shake well to disperse the spores evenly.

## Efficacy Verification of Recombinant Metarhizium Strains

### Total RNA Extraction from Fungi

1. Add **1 mL** of Buffer Rlysis-F to a 1.5 mL RNase-free microcentrifuge tube and set aside.
2. Take **20 mg** of dried hyphae, grind them into powder in liquid nitrogen, add the powder to the aforementioned 1.5 mL microcentrifuge tube, and immediately shake to mix thoroughly.
3. Add **200 μL** of chloroform to the lysed sample, mix well. Centrifuge at **12,000 rpm** at **4°C** for **5 minutes**, then collect **600 μL** of the supernatant.
4. Add **200 μL** of absolute ethanol, mix well, let stand at room temperature for **3 minutes**, centrifuge at **12,000 rpm** at **4°C** for **5 minutes**, and discard the supernatant.
5. Add **700 μL** of **75% ethanol** (prepared with DEPC-treated ddH₂O) to the precipitate for washing, centrifuge at **12,000 rpm** at **4°C** for **3 minutes**, and discard the supernatant.
6. Repeat Step 5 once.
7. Invert the tube at room temperature for **10 minutes** to air-dry the ethanol.
8. Add **50 μL** of DEPC-treated ddH₂O to dissolve the precipitate, obtaining pure RNA.

### RT-PCR Verification

Perform the reverse transcription reaction according to the following system and procedure (Table 21)

![Table 21](https://static.igem.wiki/teams/5588/experiments/fig-21-21.webp)

The reverse transcription product was used for PCR verification (Table 22)

![Table 22](https://static.igem.wiki/teams/5588/experiments/fig-22-22.webp)

[Overvoew](#bacillus-overview)
[Bacillus subtilis Construction Experiment](#bacillus-Bacillus)
[Identification of Transformants](#bacillus-Identification)
[Plasmid Transformation and Screening](#bacillus-Plasmid)
[Sucrose Induction and Metabolite Extraction](#bacillus-Sucrose)
[LC-MS Analysis](#bacillus-LC-MS)

## Detection Module – Bacillus subtilis Construction Experiment

### Activation and Cultivation of Bacterial Strains

1. Prepare LB solid medium without antibiotics. LB agar medium was purchased from Haibo Biology. Each liter contains **10 g tryptone**, **5 g yeast extract**, **5 g sodium chloride**, and **15 g agar**. For use, dissolve **35 g** of the medium in **1 L** pure water, autoclave at **121°C**, and then pour into Petri dishes to form plates.
2. The glycerol stock of Bacillus subtilis strain 168Δ4 was obtained from Biyuntian (Cat. No.: D0441). Remove the glycerol stock and thaw it on ice, then transfer it to a biosafety cabinet. All subsequent operations were performed inside the biosafety cabinet: Immerse a **200 μL** sterile pipette tip in **75% ethanol**, then flame-sterilize the tip in ethanol to ensure sterility of the tip's end. Next, use the tip to dip a small amount of the glycerol stock, and perform continuous Z-shaped streaking on the LB plate at an angle parallel to the plate. Replace with a new sterile **200 μL** pipette tip, and perform continuous Z-shaped streaking along the original streaks. Incubate the plate upside down at **37°C** overnight.
3. Prepare LB liquid medium without antibiotics. LB liquid medium was purchased from Haibo Biology. Each liter contains **10 g tryptone**, **5 g yeast extract**, and **5 g sodium chloride**. For use, dissolve **25 g** of the medium in **1 L** pure water, and autoclave at **121°C** for **15 minutes**.
4. Take the LB solid medium (without antibiotics) containing newly activated single bacterial colonies. Use tweezers to hold a sterile plastic pipette tip, pick one single colony from the plate, and place the tip (with the bacterial colony attached) into a **10 mL** test tube containing **3 mL** LB liquid medium. Incubate at **37°C** with shaking at **200 rpm** overnight.

### Identification of Transformants

1. Take the successfully transformed ***E. coli*** DH5α that was cultured overnight at **37°C**. Use a sterile pipette tip to pick single colonies from the plate obtained in the previous step, and add them to the following PCR system (Table 23). The sequences of the primers are as follows:
   * F-primer (5'-3'): GACCTCGTTTCCACCGGAAT
   * P-primer (5'-3'): TCTTCTTCCGTGATTCCTTG

![Table 23](https://static.igem.wiki/teams/5588/experiments/fig-23-23.webp)

## Plasmid Transformation and Screening

### Plasmid Transformation into Bacillus subtilis

1. Remove the prepared Bacillus subtilis competent cells from **-80°C**, thaw them on ice, then add **5 μg** of plasmid to each tube. Mix gently and incubate at **37°C** with shaking at **200 rpm** for **120 minutes**.
2. Centrifuge at **4,000×g** for **1 minute** to collect the bacterial pellet, retain **50 μL** of supernatant to resuspend the pellet, and spread the suspension onto LB solid medium containing **5 μg/mL chloramphenicol (Cmᵣ)**. Incubate the plate upside down at **37°C** overnight.

### Identification of Transformants

1. On the next day, use a sterile pipette tip to pick single colonies from the plate and inoculate them into LB liquid medium containing **5 μg/mL chloramphenicol (Cmᵣ)**. Incubate at **37°C** with shaking at **200 rpm** for **24 hours**.
2. Extract plasmids by following the protocol of the Tiangen Plasmid Extraction Kit (Cat. No.: DP103).
3. Prepare the PCR system according to the procedure in Section 7.3.2 and perform PCR.
4. To further improve the reliability of the results, conduct plasmid restriction enzyme digestion verification simultaneously. Prepare the following restriction digestion system (Table 24), then incubate the reaction mixture in a **37°C** water bath for **30 minutes**.

![Table 24](https://static.igem.wiki/teams/5588/experiments/fig-24-24.webp)

## Sucrose Induction and Metabolite Extraction

1. Inoculate the overnight-cultured Bacillus subtilis transformants into LB liquid medium containing **5 μg/mL chloramphenicol (Cmᵣ)** at a **1:100 ratio**, and incubate at **37°C** with shaking at **200 rpm** for **2 hours**.
2. For the recombinant plasmid group, add sucrose to a final concentration of **0%, 4%, 6%, and 8% (w/v)**, respectively, and incubate at **37°C** with shaking at **200 rpm** for **24 hours**.
3. After adjusting the OD₆₀₀ of all bacterial cultures to **2.0**, centrifuge at **3,000×g** for **5 minutes** at **4°C**, and place the samples on ice immediately.
4. For every **200 μL** of supernatant, add **800 μL** of acetonitrile to precipitate proteins and extract methyl salicylate.
5. Centrifuge at **21,000×g** for **3 minutes** at **4°C**; the resulting supernatant is used for LC-MS analysis.

## LC-MS Analysis

1. A Waters BEH C18 column (**1.0 mm × 50 mm, 1.7 μm**) was connected to a Dionex Ultimate 3000 system. The column oven temperature was maintained at **25°C** throughout the **5-minute** isocratic elution (**50% mobile phase B**) process.
2. The experiment was conducted at a flow rate of **100 μL/min**, using the following mobile phases: (A) **0.1% formic acid in water**; (B) **0.1% formic acid in acetonitrile**. The injection volume was set to **1 μL** for all analyses.
3. The output signal of the liquid chromatograph was connected to a Thermo Scientific Q Exactive HF mass spectrometer, which was operated in heated electrospray ionization (HESI) mode. The data acquisition duration was **5 minutes**, with parallel reaction monitoring (PRM) ion transitions configured as follows: methyl salicylate was detected in positive ion mode within **1.8–5 minutes**. The spray voltage was **3.5 kV**, the capillary temperature was set to **320°C**, the sheath gas flow rate was **35 (arb. units)**, the auxiliary gas flow rate was **10 (arb. units)**, and the maximum spray current was **100 μA**. High-frequency sampling of PRM ion transitions was performed at a resolution of **15,000**, with an automatic gain control (AGC) target value of **3×10⁶** and a maximum integration time (IT) of **500 ms**. The fixed primary mass was **50 m/z**, the isolation window width was **0.4 m/z**, and the normalized collision energy (nCE) was fixed at **35**. All data were acquired in profile mode, and peak height detection was performed using a custom Thermo Scientific Quan Browser method developed based on standard substances.

# Materials & Instruments

Materials required for the experiment: plasmid DNA, cell culture medium, transfection reagent, PCR primers, sequencing kit, etc. The main instruments include PCR instrument, centrifuge, flow cytometer, sequencer, etc.

# Materials & Instruments

## Materials List

| Name | Company | Catalog Number |
| --- | --- | --- |
| amino acid substances | Shanghai Macklin Biochemical Co., Ltd. | A810002-25g |
| Isopropyl alcohol | Macklin | I805819-500ml |
| Coomassie Brilliant Blue G-250 | Biosharp | BS044-10g |
| Ethanol anhydrous | Macklin | E803844-500ml |
| Chloroform : Isoamyl alcohol (24:1) | Macklin | C816748-100ml |
| AG RNAex Pro Reagent | Accurate Biotechnology (Hunan) Co., Ltd | AG21102 |
| PureLink RNA Mini Kit | Invitrogen, Thermo Fisher Scientific | 12183018A |
| TransScript One-Step gDNA and cDNA Synthesis SuperMix | TransGen Biotech | AT311-02 |
| PerfectStart Green qPCR SuperMix | Tiangen Biotech (Beijing) Co., Ltd | AQ101-01 |
| Tris-Glycine-SDS | Vazyme | PS105 |
| TBS/Tween Buffer 10x | Vazyme | PS103 |
| BL21 (DE3) ***Escherichia coli*** cells | TIANGEN BIOTECH (BEIJING) CO.,LTD. | CB105-02 |
| Chloramphenicol | Sangon Biotech | A600118 |
| 2 × Taq Master Mix (Dye Plus) | Vazyme | P112-01 |
| IPTG | Sangon Biotech | A600168 |
| 10x Phosphate-buffered Saline (PH 6.8) | Biosharp | BL551A |
| Protease Inhibitor Cocktail, Universal, 100× | Biosharp | BL612A |
| Lysozyme | Beyotime | P2226-5 |
| Nuclease-free Water | Biosharp | BL510A |
| Benzonase Nuclease | Beyotime | D7121-25KU |
| 0.22 μm polyethersulfone (PES) syringe filter | Biosharp | BS-PES25-22-S |
| His-tag Protein Purification Kit | Beyotime | P2226 |
| 5x SDS-PAGE Sample Loading Buffer | Biosharp | BL502B |
| Acetic acid glacial | MACKLIN | A801295 |
| Bradford Protein Assay Kit | Beyotime | P0006 |
| RNase A | Thermo Scientific™ | EN0531 |
| RiboLock RNase Inhibitor | Thermo Scientific™ | EO0381 |
| Isopropyl Alcohol | MACKLIN | I766839 |
| EDTA | Thermo Scientific™ | O2793500 |
| Ethanol [Anhydrous] | MACKLIN | E809056 |
| DL1000 DNA Marker | TAKARA | 3591Q |
| DL 500 DNA Marker | Sangon Biotech | B600303 |
| Brilliant Blue R | MACKLIN | B802269 |
| Phosphotungstic acid hydrate | Millipore | 79690-25G |
| Dual-color Prestained Low Molecular Weight Protein Ladder (1.2~40 kDa) | epizyme | WJ402 |
| Unstained Protein Molecular Weight Marker (Pierce™) | Thermo Scientific™ | 26610 |
| OCT Embedding Matrix | Sakura | 4583 |
| Ethanol, Absolute | Sinopharm Chemical Reagent Co., Ltd. | 100092683 |
| Triton® X-100 Permeabilization Solution | BioFroxx | 1139ML100 |
| Normal Goat Serum | Boster Biological Technology Co., Ltd. | AR1009 |
| TBS Buffer Salt Powder | Bioss Biotech Co., Ltd. | BP0152 |
| DAPI Solution | Solarbio | C0060 |
| Fluorescent Mounting Medium | Southern Biotech | 0100-01 |
| EGFP Antibody | Oasis biofarm | OB-PGP003 |
| LB Agar(lennox) | Qingdao Hope Bio-Technology Co., Ltd. | HB0129-2 |
| LB Broth | Qingdao Hope Bio-Technology Co., Ltd. | HB0128 |
| Agarose | Biosharp, Labgic Technology Co., Ltd. | BS081-100g |
| DNase Ⅰ, RNase-free | Thermo Fisher Scientific | EN0521 |
| DNase I | Beyotime Biotech Inc | D7073 |
| RNase T1 | Thermo Fisher Scientific | EN0541 |
| FastDigest EcoRⅠ | Thermo Fisher Scientific | FD0274 |
| Goldenview | Biosharp, Labgic Technology Co., Ltd. | BS357A |
| Superred | Biosharp, Labgic Technology Co., Ltd. | BS354B |
| T7 RNA Transcription Kit Plus | belong | SJ001V2-01 |
| FastDigest BamHⅠ | Thermo Fisher Scientific | FD0055 |
| TAE Buffer (50x) | Biosharp, Labgic Technology Co., Ltd. | BL533A |
| DNase and RNase AWAY | Beyotime Biotech Inc | B0123 |
| RNaseEraser | Foregene Co., Ltd. | RC-01011 |
| Nuclease-free Water | Biosharp, Labgic Technology Co., Ltd. | BL510B |
| Plasmid Extraction Kit (Centrifugation Column Type) | TIANGEN | DP103-03 |
| Ampicillin | biosharp | BS112 |
| Kanamycin | biosharp | BS134 |
| FastDigest NheⅠ | Thermo Fisher Scientific | FD0974 |
| FastDigest ApaⅠ | Thermo Fisher Scientific | FD1414 |
| FastDigest XbaⅠ | Thermo Fisher Scientific | FD0684 |
| DL2,000 DNA Marker | Takara | 3427Q |
| DL5,000 DNA Marker | Takara | 3428Q |
| DNA Loading Buffer (6×) | Biosharp, Labgic Technology Co., Ltd. | BL532A |
| PDA Medium | Bena Culture Collection | 370555 |
| Tween-80 | Sangon Biotech | A600562 |
| DH5α Competent Cells | Weidi Biotechnology | P815662 |
| Kanamycin Sulfate | Sangon Biotech | A506636 |
| Agrobacterium AGL1 Competent Cells | Weidi Biotechnology | AC1020 |
| Fungal Genomic DNA Extraction Kit | Sangon Biotech | B518229 |
| KH₂PO₄ | Macklin | P816382 |
| K₂HPO₄ | Macklin | M813597 |
| MgSO₄·7H₂O | Macklin | C804986 |
| CaCl₂·2H₂O | Macklin | S805275 |
| NaCl | Macklin | A801012 |
| (NH₄)₂SO₄ | Macklin | F809847 |
| FeSO₄·7H₂O | Macklin | A601111 |
| Acetosyringone | Sangon Biotech | A100169 |
| MES | Sangon Biotech | B0394 |
| H₃BO₃ | sigma | A500331 |
| MnCl₂·4H₂O | Sangon Biotech | 767420 |
| Na₂MoO₄·2H₂O | Macklin | Z820755 |
| ZnCl₂ | Macklin | I809489 |
| FeCl₃·6H₂O | Macklin | C805353 |
| CuSO₄·5H₂O | Macklin | S818055 |
| Na₂SO₄ | Macklin | P816348 |
| KCl | Macklin | C805758 |
| CaCl₂, anhydrous | Macklin | 221295 |
| KNO₃ | sigma | A362623 |
| Glufosinate | Sangon Biotech | F556549 |
| Cefotaxime sodium | Sangon Biotech | A430167 |
| Peptone | Macklin | P914715 |
| Dimethyl sulfoxide | Macklin | D806645 |
| Sterile Filter Membrane | Huankai | 21624 |
| 75% Ethanol | Macklin | E809056 |
| IM Induction Medium (Liquid) | Ararat | 58242 |
| IM Induction Medium (Solid) | Ararat | 69261 |
| Fungal Total RNA Rapid Extraction Kit | Sangon Biotech | B518629 |
| Bacillus subtilis 168Δ4 Glycerol Stock | Beyotime | D0441 |
| Methyl Salicylate | Shanghai Macklin Biochemical Co., Ltd. | M816819 |
| Acetonitrile | Macklin | A805878 |
| Formic Acid | Macklin | F805088 |
| Sucrose | Macklin | S814888 |
| GM Ⅰ Medium, GM Ⅱ Medium | Chinook | GM-001 |
| LB agar medium | Qingdao Hope Bio-Technology Co., Ltd. | GM-002 |
| LB broth medium | Qingdao Hope Bio-Technology Co., Ltd. | HB0128 |
| Chloramphenicol | Sangon Biotech | HB0129 |
| Ampicillin | Sangon Biotech | C602545 |
| CaCl₂ | Chinook | A600199 |
| MgCl₂ | Chinook | C1001 |
| BamHI | Takara | M1002 |
| ApaⅠ | Takara | 1610A |
| Fast digest buffer | Takara | 1041A |

## Instruments

| Instrument / Equipment Name | Company / Manufacturer |
| --- | --- |
| QuantStudio 5 Real-Time PCR System | Thermo Fisher Scientific |
| Biometra TAdvanced (PCR System) | Analytik Jena |
| Precision Balance, 320g Capacity, 1mg Readability, BSA323S-CW | Sartorius Thailand Co., Ltd. |
| Centrifuge 5430 - Microcentrifuge | EPPENDORF CO., LTD. |
| Centrifuge 5418 R (Refrigerated Centrifuge) | EPPENDORF CO., LTD. |
| D1008 Low Speed Centrifuge (Mini Centrifuge) | DLAB SCIENTIFIC CO., LTD. |
| NanoPhotometer N50 (Ultramicrospectrophotometer) | Implen GmbH |
| HYK Dry Bath (Constant Temperature Metal Bath HYK0032) | HYK Huayinkang |
| UVP Transilluminator (Benchtop UV Transilluminator) | Analytik Jena GmbH + Co. KG |
| Electrically Heated Constant Temperature Incubator | SHANGHAI BLUEPARD INSTRUMENTS CO., LTD. |
| Isotherm® Forced Convection Lab Oven | Esco |
| Steam Sterilizer (Laboratory Use, Large Capacity, Vertical Autoclave) | YAMATO SCIENTIFIC CO., LTD. |
| Benchtop Incubator Shaker (Constant Temperature Oscillating Incubator ZQTY-70N) | Shanghai Zhichu General Equipment Development Co., Ltd. |
| Ultrasonic Homogenizer JY92-IIN (Ultrasonic Cell Disruptor) | Ningbo Scientz Biotechnology Co., Ltd. |
| Drill (Drilling Machine) | DELIXI GROUP CO., LTD. |
| Zeiss Axioplan II Fluorescence Microscope | Zeiss |
| Transference Decoloring Shaker (Side-to-Side Shaker) | Beyotime |
| Multiskan FC Microplate Photometer | Thermo Scientific™ |
| Mini-PROTEAN® Tetra Cell System (Vertical Electrophoresis Cell) | BIO-RAD |
| Cryostat (Frozen Section Machine) | Thermo Fisher Scientific |
| Anti-Drop Microscope Slides | Bioss Biotech Co., Ltd. |
| Fluorescence Microscope | Olympus Corporation |
| Imaging System | 3D HISTECH Ltd. |
| Pipette Gun (Micropipette) | DLAB Scientific Co., Ltd. |
| Histochemical Pen | Fuzhou Maixin Biotech Co., Ltd. |
| LC System | Dionex Ultimate 3000 |
| Column | Waters ACQUITY BEH C18, 1.0 mm × 50 mm, 1.7 µm |
| Mass Spectrometer (MS) | Thermo Scientific Q Exactive HF with HESI source |

# Sequences

The experimental procedure includes the following steps: cell culture, plasmid construction, gene transfection, gene editing, cell screening, sequencing analysis, data processing, and other steps.

# Sequence

In this section, we have systematically organized the sequences into the following 5 modules. All sequences mentioned in our project can be found in these 5 modules.

## RNA Molecules with Silencing Function

In this module, you can find all RNA molecule sequences with silencing function. You can quickly locate the sequence you need based on the name of the RNA or the name of the target it silences.

| Name | Application | Sequence (5'→3') |
| --- | --- | --- |
| dsCHS | Single-target dsRNA for silencing the CHS gene in brown citrus aphid | UUGAUACCUCUCAAACGUUGUAGGUGUUUGACGAUAACGACUGCUUGUUUGUCUAGUAGACCAUCGUGGGUCAAAUCUUCAGUCUUUUUGUUGCAACACAAGUUUAAGUCCGUCGAAGCAAGUAUGUGAGACAAAGUACCGAAUCGAUGGAACAACAUGGCGGUAAACUGAACUACUAUGAUGAACGCGAAGAAGAACACAAACACCAAACCAAUGGGUUCCAAGUGUAAGUACUCUUUGCUGAUAAUGAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGC |
| dsCP | Single-target dsRNA for silencing the CP19 gene in brown citrus aphid | UUAAAAGUCGAACGCGUCGUUGCUGGGCUUCCUGUAUCCUGUGUUUCCGCUAUCGGACCGGCUAACCACCGCGUUGAAACCUGUUUCUGGGUCUGCGGUGUACUCGACCAGCCUCAUGGCGCCAUCGGGUUCCAGAACACUGUACGCUCCCUUGACCACAUCACCCUCUCGCGCUUCCCAUUGGCUCUUCACGUCGUAAGUUUCCGGAUCGUGAACCGCGUAAUCGAACUUGUACGCUUUGGGUGCGUAAUUUUCUGCAGACUCUGAGCUUUGAGAUCGGGGUCCUCCUUCAACGAAAAUCGGUAUGGCCAUGGUGCAUGCCGUCAUUACGCUCAGCGCUGCUAAAAUUUGACACGACCUCAU |
| dsCYP450 | Single-target dsRNA for silencing the CYP450 gene in brown citrus aphid | AACAAAGUGUCAACAGUCAUUCCAAUUACGUCUUUGUAAUCUGCCUCGUCUGAGCUCAAGUAUUCUCCUAAUAAUGAUGCUUUGGAGUCAGUCGUUUUAAUACUGGUUAGUUUUUCACUUACAACUUUUAGGGCCAUUUUUUCAAUGUAUUCGUGUCCUUUUUUUAUACUUUUAUACAUGGGGGUAUCAAAUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUUCAUCGAAGGC |
| **dsF2** | Dual-target fusion dsRNA for silencing CHS and CYP450 genes in brown citrus aphid | AGCUAACAAAGUGUCAACAGUCAUUCCAAUUACGUCUUUGUAAUCUGCCUCGUCUGAGCUCUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGCUCAUCGAUCGGAUAUAAAUAUUUGUCUAUCAGAUCUCGCCAGAACUGUUGUUUCGGAGUAUCGACUUUAACAUUUCCAUCAUUUCUUCGUGGGUUUCGUGCCACAUGGUCGCCACAGAUAUUGUUUCAUAAUAUUCGUCUGGUUCUUUAUCUCGAUCGGCCAA |
| **dsF3** | Triple-target fusion dsRNA for synergistic silencing of CHS, CYP450 and CP19 genes in brown citrus aphid | GUUGCUGGGCUUCCUGUAUCCUGUGUUUCCGCUAUCGGACCGGCUAACCAUUUUCUCCAUAGUUGUGGUCCAUUAUCAGUCUUCAGUAUCGCACUGUUUAUAUCGUGAGCUGCUUUGAUCAAUUUCGAACUUUCCGAAUUAGAGUCCCAUUCUUCGUCUUUGAACCACUGUAGUCGUAUCUCAGACGUCUCUGCAAUAGUCGUAAUAUUUGUCUUGACGCCUAUCGGCUCAUCGAUCGGAUAUAAAUAUUUGUCUAUCAGAUCUCGCCAGAACUGUUGUUUCGGAGUAUCGACUUUAACAUUUCCAUCAUUUCUUCGUGGGUUUCGUGCCACAUGGUCGCCACAGAUAUUGUUUCAUAAUAUUCGUCUGGUUCUUUAUCUCGAUCGGCCAA |
| tri-shRNA | Triple tandem shRNA for combined silencing of CHS, CYP450 and CP19 genes in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) | **ACAUGGGUAACCUCAUGU** UCUAGA CGCCGUCGAACAAUUUACAACUUAG UUCAAGAGA CUAAGUUGUAAAUUGUUCGACGGCG AGGCAU GAGCUCAGACGAGGCAGAUUACAAA UUCAAGAGA UUUGUAAUCUGCCUCGUCUGAGCUC GAAUUC GAGCGUACAGUGUUCUGGAACCCGA UUCAAGAGA UCGGGUUCCAGAACACUGUACGCUC |
| bi-amiRNA | Double tandem amiRNA for combined silencing of CHS and CYP450 genes in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) | **ACAUGAGGUUACCCAUGU** AGUUCU CUCGAGUCUGCUCCGUCUAAUGUUU CGGUAUAUUUAAGAU AAACAUUAGACGGAGGAGACUCGAG UG UCCGUA UUCAACAUUUAUCAAGCUGCGGC GCUGGCGCCUGC GCGGCAGCUUGUUAAAUGUUGAAUC |
| amiRNA | amiRNA for silencing the CHS gene in brown citrus aphid (bold sequence is pac site, underlined sequence is the final siRNA that exerts silencing effect) | gu cgccgucgaucaauuuacaacuuag uagaauuuauauggc cuaaguuguaaauuguucgacggcg ucuuga **acauggguaauccucauguuu** |

## Plasmid Sequences

In this module, you can find the sequences of plasmids constructed throughout the entire project. You can quickly locate the sequence you are looking for based on the plasmid's name or its function.

| Plasmid Name | Function | Complete Vector Sequence (5'→3') |
| --- | --- | --- |
| pUC57\_pac-bi-amiRNA | DNA sequence for producing bi-amiRNA molecules (bold sequence is pac site, underlined sequence is target) | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagctaatacgactcactataggccacagcatacatgggtaacctcatgttcaagagagctcagacgaggcagattacaaagccatataaattctatttgtaatctgcctcctctgagctcacaggcataagttgtaaatagttcgacgccgcgaccgcggacgcgccgtcgaacaatttacaacttagggaagagcggatcctctagagggcccgtttaaacccgctgatcagcctcgactctagcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagctcagatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaagcccaatctgaataatgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgaggcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtttttccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca |
| pUC57\_mini-Kana-BsmBI⁻-terminator-ΔT7 | Serves as the basic original cloning backbone, with subsequent insertion of corresponding target fragments | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactCTAGCTTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCATTACTGGATCTATCAACAGGAGTCCAAGCTCAGatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaagcccaatctgaataatgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgaggcgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtttttccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaagcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca |
| pUC57\_pac-tri-shRNA | DNA sequence for producing tri-shRNA (CHS+CYP450+CP) (bold sequence is pac site, underlined sequence is target) | tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggacgtcattgtcgatctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctctctggctaactagagaacccactgcttactggcttatcgaaatgctagctaatacgactcactataggagcatacatgggtaacctcatgttctagacgccgtcgaacaatttacaacttagttcaagagactaagttgtaaattgttcgacggcgaggcatgagctcagacgaggcagattacaaattcaagagatttgtaatctgcctcgtctgagctcgaattcgagcgtacagtgttctggaacccgattcaagagatcgggttccagaacactgtacgctcccacggaagagcggatcctctagagggcccgtttaaacccgctgatcagcctcgactctagcttggattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtccaagctcagatcattgtcacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcaggatcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacgggccagagctgca |
| pACYCDuet\_1-CP-Histag-CP | Can express His-tagged MS2 capsid protein dimer fusion protein | GGGGAATTGTGAGCGGATAACAATTCCCCTGTAGAAATAATTTTGTTTAACTTTAATAAGGAGATATACCATGGCTTCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGAACTGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACGCTAACTTTACTCAGTTCGTTCTCGTCGACAATGGCGGTACCCATCACCATCACCATCACGGTACCGGCGACGTGACTGTCGCCCCAAGCAACTTCGCTAACGGGGTCGCTGAATGGATCAGCTCTAACTCGCGTTCACAGGCTTACAAAGTAACCTGTAGCGTTCGTCAGAGCTCTGCGCAGAATCGCAAATACACCATCAAAGTCGAGGTGCCTAAAGTGGCAACCCAGACTGTTGGTGGTGTAGAGCTTCCTGTAGCCGCATGGCGTTCGTACTTAAATATGGAACTAACCATTCCAATTTTCGCTACGAATTCCGACTGCGAGCTTATTGTTAAGGCAATGCAAGGTCTCCTAAAAGATGGAAACCCGATTCCCTCAGCAATCGCAGCAAACTCCGGCATCTACTAATAGACGCCGGCCAgcGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTATTGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCAGATCTCAATTGGATATCGGCCGGCCACGCGATCGCTGACGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGGTAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACCGGGTCGAATTTGCTTTCGAATTTCTGCCATTCATCCGCTTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAGGGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAGACGGCATGATGAACCTGAATCGCCAGCGGCATCAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATAGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCAAAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTGCGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTACTGATTTAGTGTATGATGGTGTTTTTGAGGTGCTCCAGTGGCTTCTGTTTCTATCAGCTGTCCCTCCTGTTCAGCTACTGACGGGGTGGTGCGTAACGGCAAAAGCACCGCCGGACATCAGCGCTAGCGGAGTGTATACTGGCTTACTATGTTGGCACTGATGAGGGTGTCAGTGAAGTGCTTCATGTGGCAGGAGAAAAAAGGCTGCACCGGTGCGTCAGCAGAATATGTGATACAGGATATATTCCGCTTCCTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGAAATGGCTTACGAACGGGGCGGAGATTTCCTGGAAGATGCCAGGAAGATACTTAACAGGGAAGTGAGAGGGCCGCGGCAAAGCCGTTTTTCCATAGGCTCCGCCCCCCTGACAAGCATCACGAAATCTGACGCTCAAATCAGTGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCTGGCGGCTCCCTCGTGCGCTCTCCTGTTCCTGCCTTTCGGTTTACCGGTGTCATTCCGCTGTTATGGCCGCGTTTGTCTCATTCCACGCCTGACACTCAGTTCCGGGTAGGCAGTTCGCTCCAAGCTGGACTGTATGCACGAACCCCCCGTTCAGTCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGAAAGACATGCAAAAGCACCACTGGCAGCAGCCACTGGTAATTGATTTAGAGGAGTTAGTCTTGAAGTCATGCGCCGGTTAAGGCTAAACTGAAAGGACAAGTTTTGGTGACTGCGCTCCTCCAAGCCAGTTACCTCGGTTCAAAGAGTTGGTAGCTCAGAGAACCTTCGAAAAACCGCCCTGCAAGGCGGTTTTTTCGTTTTCAGAGCAAGAGATTACGCGCAGACCAAAACGATCTCAAGAAGATCATCTTATTAATCAGATAAAATATTTCTAGATTTCAGTGCAATTTATCTCTTCAAATGTAGCACCTGAAGTCAGCCCCATACGATATAAGTTGTAATTCTCATGTTAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATGTCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAATTAATACGACTCACTATA |
| pET28a\_CP-GBP3.1-CP-TAT-Histag | Can express His-tagged single-chain capsid protein dimer fused with GBP3.1 (aphid intestinal targeting peptide) and TAT (cell-penetrating peptide) | TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGATCCGCTAGTAACTTCACCCAATTTGTTCTGGTTGATAATGGCGGCACCGGTGACGTAACCGTGGCTCCGTCTAACTTCGCGAACGGCGTCGCGGAATGGATTTCCAGCAACAGCCGCTCGCAAGCGTATAAGGTGACGTGCAGCGTTCGTCAGAGCTCGGCTCAGAACCGCAAGTACACGATCAAGGTCGAGGTTCCGAAAGTGGCGACGCAAACCGTTGGTGGCGTGGAGCTGCCGGTCGCTGCTTGGCGCAGCTATCTGAACATGGAATTGACCATCCCGATCTTTGCGACCAATTCCGACTGTGAACTGATTGTGAAAGCAATGCAGGGCTTGCTGAAGGACGGCAACCCGATCCCGAGCGCGATCGCAGCGAATTCTGGTATTTACACCTGCAGCAAAAAATACCCGCGTTCCCCGTGTATGGGTGACGTGACTGTAGCGCCAAGCAACTTTGCCAACGGTGTCGCTGAATGGATCAGCTCTAACAGTCGCTCCCAGGCATACAAGGTGACTTGCTCCGTGCGTCAAAGCAGCGCCCAAAACCGTAAATATACCATTAAGGTGGAGGTGCCGAAGGTTGCGACCCAGACCGTTGGTGGCGTTGAGCTCCCTGTTGCCGCGTGGCGTAGCTACCTGAATATGGAATTGACCATTCCGATCTTCGCGACGAACAGCGATTGCGAGTTAATTGTTAAAGCCATGCAGGGTCTGCTGAAAGATGGTAATCCGATCCCATCTGCCATCGCCGCAAATTCCGGCATTTACCAGCTGCAGGGCGGTCCGGCACCGGGTGGTAGCGCGTATGGTCGTAAGAAACGTCGCCAACGTCGTAGACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT |
| pACYCDuet\_1-CP-Histag-CP-shRNA | Can express intact His-tagged virus-like particles (VLPs) that encapsulate shRNA with a pac site | ggggaattgtgagcggataacaattcccctgtagaaataattttgtttaactttaataaggagatataccatggcttctaactttactcagttcgttctcgtcgacaatggcggaactggcgacgtgactgtcgccccaagcaacttcgctaacggggtcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaagtggcaacccagactgttggtggtgtagagcttcctgtagccgcatggcgttcgtacttaaatatggaactaaccattccaattttcgctacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctacgctaactttactcagttcgttctcgtcgacaatggcggtacccatcaccatcaccatcacggtaccggcgacgtgactgtcgccccaagcaacttcgctaacggggtcgctgaatggatcagctctaactcgcgttcacaggcttacaaagtaacctgtagcgttcgtcagagctctgcgcagaatcgcaaatacaccatcaaagtcgaggtgcctaaagtggcaacccagactgttggtggtgtagagcttcctgtagccgcatggcgttcgtacttaaatatggaactaaccattccaattttcgctacgaattccgactgcgagcttattgttaaggcaatgcaaggtctcctaaaagatggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccagcggccgcataatgcttaagtcgaacagaaagtaatcgtattgtacacggccgcataatcgaaattaatacgactcactataggggcagcggcagctagttaaatgttgaatcatcttaaatataccggattcaacatttaacaagctgccgcagaacttgtacccattaggagtacaaacctaggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaacctcaggcatttgagaagcacacggtcacactgcttccggtagtcaataaaccggtaaaccagcaatagacataagcggctatttaacgaccctgccctgaaccgacgaccgggtcgaatttgctttcgaatttctgccattcatccgcttattatcacttattcaggcgtagcaccaggcgtttaagggcaccaataactgccttaaaaaaattacgccccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccgacatggaagccatcacagacggcatgatgaacctgaatcgccagcggcatcagcaccttgtcgccttgcgtataatatttgcccatagtgaaaacgggggcgaagaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccagggattggctgagacgaaaaacatattctcaataaaccctttagggaaataggccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactgccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgctcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcaccgtctttcattgccatacggaactccggatgagcattcatcaggcgggcaagaatgtgaataaaggccggataaaacttgtgcttatttttctttacggtctttaaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaactgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaacggtggtatatccagtgatttttttctccattttagcttccttagctcctgaaaatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggtgaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagttggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcgaagtgatcttccgtcacaggtatttattcggcgcaaagtgcgtcgggtgatgctgccaacttactgatttagtgtatgatggtgtttttgaggtgctccagtggcttctgtttctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacggcaaaagcaccgccggacatcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttcccctggcggctccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatataagttgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaaattaatacgactcactata |
| pET28a\_GBP3.1-EGFP-TAT-Histag | Can express His-tagged enhanced green fluorescent protein (EGFP) fused with GBP3.1 (aphid intestinal targeting peptide) and TAT (cell-penetrating peptide) | TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCACATGTTCAAAAAAGTATCCCAGGTCTCCGTGTATGGGTGGCGGTGGATCTGGTGGTGGTGGCTCAGGTGGTGGCGGCTCCGGTGGTGGTGGCAGCATGGTGTCTTTTAAGAGCCTGTTGGTGCTGTGTTGCGCGGCGCTGGGCGCATTCGCGACCAAACGTATGAGCAAAGGCGAGGAATTGTTTACGGGTGTCGTGCCAATTCTGGTGGAACTGGATGGCGACGTTAACGGCCACAAGTTCTCCGTTAGCGGTGAGGGCGAGGGCGACGCCACCTACGGTAAATTGACCTTAAAATTCATCTGCACCACCGGTAAGCTGCCGGTTCCGTGGCCTACATTGGTAACGACGTTTACGTACGGCGTTCAATGCTTTAGCCGTTATCCGGATCACATGAAACGCCACGATTTTTTCAAGTCTGCTATGCCGGAAGGCTATGTTCAAGAGCGCACCATTTTCTTTAAGGACGACGGCAACTACAAAACCCGTGCGGAAGTGAAATTCGAGGGTGACACCCTGGTGAATCGTATTGAACTCAAGGGTATTGATTTCAAGGAGGACGGTAACATTCTGGGTCATAAGCTGGAGTATAACTACAACAGCCATAATGTCTATATCATGGCAGATAAGCAGAAAAACGGTATCAAGGCTAATTTTAAGACCAGACACAACATCGAAGATGGCGGAGTCCAGCTGGCGGACCACTACCAGCAGAACACTCCGATCGGCGACGGCCCAGTTCTGTTACCGGATAATCATTACCTTAGCACCCAAAGCGCGCTGAGCAAGGACCCGAATGAAAAACGTGATCACATGGTTCTGTTGGAGTTCGTGACCGCTGCCGGTATCACCCACGGTATGGACGAATTGTATAAACTGCAAGGTGGTCCGGCACCGGGTGGCTCGGCGTACGGCCGTAAAAAACGCCGTCAGCGTCGCCGGCTGCAAGGTGGTCCGGCACCGGGTGGCTCGGCGCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT |
| pET28a\_EGFP-Histag | Can express His-tagged enhanced green fluorescent protein (EGFP) | TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCCTCCAGGGTGGTCCGGCACCGGGTGGCAGCGCTATGGTGAGCTTTAAGTCGCTGCTGGTCTTATGCTGCGCCGCGCTGGGCGCATTCGCGACCAAACGTATGAGCAAGGGTGAGGAACTGTTTACCGGCGTGGTTCCGATCCTGGTTGAATTAGATGGCGACGTGAACGGTCACAAGTTCTCTGTTTCTGGTGAGGGTGAAGGAGACGCCACCTACGGGAAATTGACCCTTAAGTTCATCTGCACCACCGGTAAATTGCCGGTTCCGTGGCCTACCTTGGTAACAACGTTTACGTACGGTGTTCAATGTTTTAGCCGTTATCCGGACCACATGAAACGCCACGATTTCTTCAAGTCCGCTATGCCGGAAGGCTATGTGCAAGAGCGCACCATTTTTTTCAAGGACGACGGCAACTACAAGACTCGCGCGGAAGTTAAATTCGAGGGTGACACCCTGGTGAATCGTATTGAGCTGAAAGGTATTGATTTTAAAGAAGATGGAAATATTCTGGGTCATAAGCTGGAGTACAACTATAACAGCCATAATGTTTATATTATGGCAGATAAACAGAAAAACGGCATCAAAGCGAACTTTAAGACGCGTCATAACATCGAGGACGGCGGTGTCCAGCTGGCGGATCACTACCAGCAAAACACCCCGATCGGCGACGGCCCAGTGTTGCTGCCGGATAATCATTACCTGTCCACCCAGAGCGCTCTGTCCAAGGACCCGAATGAAAAACGTGATCACATGGTTCTGCTTGAGTTCGTGACTGCCGCGGGTATCACCCACGGCATGGATGAACTGTATAAGCTCCAGGGTGGTCCGGCACCGGGTGGCAGCGCTCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT |
| pUC57\_CHS | For producing double-stranded RNA (dsRNA) of the CHS gene (The underlined sequence is the target sequence) | TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATCATTATCAGCAAAGAGTACTTACACTTGGAACCCATTGGTTTGGTGTTTGTGTTCTTCTTCGCGTTCATCATAGTAGTTCAGTTTACCGCCATGTTGTTCCATCGATTCGGTACTTTGTCTCACATACTTGCTTCGACGGACTTAAACTTGTGTTGCAACAAAAAGACTGAAGATTTGACCCACGATGGTCTACTAGACAAACAAGCAGTCGTTATCGTCAAACACCTACAACGTTTGAGAGGTATCAACCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC |
| pUC57\_CHS-tri-terminator | For producing double-stranded RNA (dsRNA) of the CHS gene (The underlined sequence is the target sequence) | TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGGTCGACCAGCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGAAGCTTCTTTCAGCAAAAAACCCCGCGAGACCCCCGAAGAGGCCCCGCGGGGTTATGCTAGTAATACGACTCACTATAGGGGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATCATTATCAGCAAAGAGTACTTACACTTGGAACCCATTGGTTTGGTGTTTGTGTTCTTCTTCGCGTTCATCATAGTAGTTCAGTTTACCGCCATGTTGTTCCATCGATTCGGTACTTTGTCTCACATACTTGCTTCGACGGACTTAAACTTGTGTTGCAACAAAAAGACTGAAGATTTGACCCACGATGGTCTACTAGACAAACAAGCAGTCGTTATCGTCAAACACCTACAACGTTTGAGAGGTATCAACCCTATAGTGAGTCGTATTACTAGCATAACCCCGCGGGGCCTCTTCGGGGGTCTCGCGGGGTTTTTTGCTGAAAGAAGCTTCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGCTGGTCGACCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC |
| pUC57\_dsF2 | For producing double-stranded RNA (dsRNA) of the dual fusion gene (The underlined sequence is the target sequence) | TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTTGGCCGATCGAGATAAAGAACCAGACGAATATTATGAAACAATATCTGTGGCGACCATGTGGCACGAAACCCACGAAGAAATGATGGAAATGTTAAAGTCGATACTCCGAAACAACAGTTCTGGCGAGATCTGATAGACAAATATTTATATCCGATCGATGAGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATACGACTACAGTGGTTCAAAGACGAAGAATGGGACTCTAATTCGGAAAGTTCGAAATTGATCAAAGCAGCTCACGATATAAACAGTGCGATACTGAAGACTGATAATGGACCACAACTATGGAGAAAAGAGCTCAGACGAGGCAGATTACAAAGACGTAATTGGAATGACTGTTGACACTTTGTTAGCTCCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC |
| pUC57\_dsF3 | For producing dsF3 molecules (The underlined sequence is the target sequence) | TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTCTAATACGACTCACTATAGGGTTGGCCGATCGAGATAAAGAACCAGACGAATATTATGAAACAATATCTGTGGCGACCATGTGGCACGAAACCCACGAAGAAATGATGGAAATGTTAAAGTCGATACTCCGAAACAACAGTTCTGGCGAGATCTGATAGACAAATATTTATATCCGATCGATGAGCCGATAGGCGTCAAGACAAATATTACGACTATTGCAGAGACGTCTGAGATACGACTACAGTGGTTCAAAGACGAAGAATGGGACTCTAATTCGGAAAGTTCGAAATTGATCAAAGCAGCTCACGATATAAACAGTGCGATACTGAAGACTGATAATGGACCACAACTATGGAGAAAATGGTTAGCCGGTCCGATAGCGGAAACACAGGATACAGGAAGCCCAGCAACCCCTATAGTGAGTCGTATTAGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC |
| pBar\_pac-dsF3 | For modifying Metarhizium anisopliae to produce dsF3 molecules (The bold sequence is the pac site, and the underlined sequence is the target sequence) | cccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcaggatcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatctggatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattgctctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacatgattacgaattccactagtgtcgacagaagatgacattgaaggagcactttttgggcttggctggagctagtggaggtcaacaatgaatgcctattttggtttagtcgtccaggcggtgagcacaaaatttgtgtcgtttgacaagatggttcatttaggcaactggtcagatcagccccacttgtagcagtagcggcggcgctcgaagtgtgactcttattagcagacaggaacgaggacattattatcatctgctgctggtgcacgataacttggtgcgtttgtcaagcaaggtaagtgaacgacccggtcataccttcttaagttcgcccttcctccctttatttcagattcaatctgacttacctattctacccaagcatcgataagcggccgctctagtggatctaccatgagcccagaacgacgcccggccgacatccgccgtgccaccgaggcggacatgccggcggtctgcaccatcgtcaaccactacatcgagacaagcacggtcaacttccgtaccgagccgcaggaaccgcaggagtggacggacgacctcgtccgtctgcgggagcgctatccctggctcgtcgccgaggtggacggcgaggtcgccggcatcgcctacgcgggcccctggaaggcacgcaacgcctacgactggacggccgagtcgaccgtgtacgtctccccccgccaccagcggacgggactgggctccacgctctacacccacctgctgaagtccctggaggcacagggcttcaagagcgtggtcgctgtcatcgggctgcccaacgacccgagcgtgcgcatgcacgaggcgctcggatatgccccccgcggcatgctgcgggcggccggcttcaagcacgggaactggcatgacgtgggtttctggcagctggacttcagcctgccggtaccgccccgtccggtcctgcccgtcaccgaaatctgaggatccacttaacgttactgaaatcatcaaacagtttgacgaatctggatataagatcgttggtgtcgatgtcagctccggagttgagacaaatggtgttcaggatctcgataagatacgttcatttgtccaagcagcaaagagtgccttctagtgatttaatagctccatgtcaacaagaataaaacgcgtttcgggtttacctcttccagatacagctcatctgcaatgcattaatgcattggacctcgcaaccctagtacgcccttcaggctccggcgaagcagaagaatagcttagcagagtctattttcattttcgggagacgagatcaagcagatcaacggtcgtcaagagacctacgagactgaggaatccgctcttggctccacgcgactatatatttgtctctaattgtactttgacatgctcctcttctttactctgatagcttgactatgaaaattccgtcaccagcccctgggttcgcaaagataattgcactgtttcttccttgaactctcaagcctacaggacacacattcatcgtaggtataaacctcgaaaatcattcctactaagatgggtatacaatagtaaccatgcatggttgcctagtgaatgctccgtaacacccaatacgccggccgaaacttttttacaactctcctatgagtcgtttacccagaatgcacaggtacacttgtttagaggtaatccttctttctagagtcgacctgcagactagtgtcgacagaagatgacattgaaggagcactttttgggcttggctggagctagtggaggtcaacaatgaatgcctattttggtttagtcgtccaggcggtgagcacaaaatttgtgtcgtttgacaagatggttcatttaggcaactggtcagatcagccccacttgtagcagtagcggcggcgctcgaagtgtgactcttattagcagacaggaacgaggacattattatcatctgctgcttggtgcacgataacttggtgcgtttgtcaagcaaggtaagtgaacgacccggtcataccttcttaagttcgcccttcctccctttatttcagattcaatctgacttacctattctacccaagcatcgataattggcgcgccacatgggtaacctcatgtttggccgatcgagataaagaaccagacgaatattatgaaacaatatctgtggcgaccatgtggcacgaaacccacgaagaaatgatggaaatgttaaagtcgatactccgaaacaacagttctggcgagatctgatagacaaatatttatatccgatcgatgagccgataggcgtcaagacaaatattacgactattgcagagacgtctgagatacgactacagtggttcaaagacgaagaatgggactctaattcggaaagttcgaaattgatcaaagcagctcacgatataaacagtgcgatactgaagactgataatggaccacaactatggagaaaatggttagccggtccgatagcggaaacacaggatacaggaagcccagcaacttcaagagacaacgacccgaaggacataggacacaaaggcgatagcctggccgattggtaaaagaggtatcaacaccaggtaatagtcagaagtcatagcgtgacaaatatagcactcgacgaaactagttaaagcttgaaaggcttaatctcagggtaagaagcagaaacttggtgacatcagcatagagtctgcagagacgttatcagcattataaacagaactgcggatagccgagtagctagcctatatttataaacagatagtctagagcggtcttgacaacaaagcctcatagctgaaattgtaaaggtagtaaagaagcacccaaagcacggtgtaccagcggtgtctataacaaagtattataagcagaccaagaaatagagctagccggttctcgagacttaacgttactgaaatcatcaaacagtttgacgaatctggatataagatcgttggtgtcgatgtcagctccggagttgagacaaatggtgttcaggatctcgataagatacgttcatttgtccaagcagcaaagagtgccttctagtgatttaatagctccatgtcaacaagaataaaacgcgtttcgggtttacctcttccagatacagctcatctgcaatgcattaatgcattggacctcgcaaccctagtacgcccttcaggctccggcgaagcagaagaatagcttagcagagtctattttcattttcgggagacgagatcaagcagatcaacggtcgtcaagagacctacgagactgaggaatccgctcttggctccacgcgactatatatttgtctctaattgtactttgacatgctcctcttctttactctgatagcttgactatgaaaattccgtcaccagcccctgggttcgcaaagataattgcactgtttcttccttgaactctcaagcctacaggacacacattcatcgtaggtataaacctcgaaaatcattcctactaagatgggtatacaatagtaaccatgcatggttgcctagtgaatgctccgtaacacccaatacgccggccgaaacttttttacaactctcctatgagtcgtttacccagaatgcacaggtacacttgtttagaggtaatccttcttaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgagcttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttaaagtactttaaagtactttaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgagcggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaacttattaaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccc |
| pgrac100 | Bacillus subtilis-Escherichia coli shuttle plasmid, serving as the original plasmid backbone | ttaagttattggtatgactggttttaagcgcaaaaaaagttgctttttcgtacctattaatgtatcgttttagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcaggccttaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgttttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaattagcttggtaccaAAGGAGGTAAGGATCACTAGAAAATTTTTTAAAAAATCTCTTGACATTGGAAGGGAGATATGTTATTATAAGAATTGCGGAATTGTGAGCGGATAACAATTCCCATATAAAGGAGGAAGGATCAATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAGCATCAGCTGCTGGATCCATGTCTAGACTGCAGACTAGTGTCGACGTCGCGGCCGCACATCACCATCACCATCACCATCACTAACGTCCCCggggcagcccgcctaatgagcgggcttttttcacgtcacgcgtccatggagatctttgtctgcaactgaaaagtttataccttacctggaacaaatggttgaaacatacgaggctaatatcggcttattaggaatagtccctgtactaataaaatcaggtggatcagttgatcagtatattttggacgaagctcggaaagaatttggagatgacttgcttaattccacaattaaattaagggaaagaataaagcgatttgatgttcaaggaatcacggaagaagatactcatgataaagaagctctaaaactattcaataaccttacaatggaattgatcgaaagggtggaaggttaatggtacgaaaattaggggatctacctagaaagccacaaggcgataggtcaagcttaaagaacccttacatggatcttacagattctgaaagtaaagaaacaacagaggttaaacaaacagaaccaaaaagaaaaaaagcattgttgaaaacaatgaaagttgatgtttcaatccataataagattaaatcgctgcacgaaattctggcagcatccgaagggaattcatattacttagaggatactattgagagagctattgataagatggttgagacattacctgagagccaaaaaactttttatgaatatgaattaaaaaaaagaaccaacaaaggctgagacagactccaaacgagtctgtttttttaaaaaaaatattaggagcattgaatatatattagagaattaagaaagacatgggaataaaaatattttaaatccagtaaaaatatgataagattatttcagaatatgaagaactctgtttgtttttgatgaaaaaacaaacaaaaaaaatccacctaacggaatctcaatttaactaacagcggccaaactgagaagttaaatttgagaaggggaaaaggcggatttatacttgtatttaactatctccattttaacattttattaaaccccatacaagtgaaaatcctcttttacactgttcctttaggtgatcgcggagggacattatgagtgaagtaaacctaaaaggaaatacagatgaattagtgtattatcgacagcaaaccactggaaataaaatcgccaggaagagaatcaaaaaagggaaagaagaagtttattatgttgctgaaacggaagagaagatatggacagaagagcaaataaaaaacttttctttagacaaatttggtacgcatataccttacatagaaggtcattatacaatcttaaataattacttctttgatttttggggctattttttaggtgctgaaggaattgcgctctatgctcacctaactcgttatgcatacggcagcaaagacttttgctttcctagtctacaaacaatcgctaaaaaaatggacaagactcctgttacagttagaggctacttgaaactgcttgaaaggtacggttttatttggaaggtaaacgtccgtaataaaaccaaggataacacagaggaatccccgatttttaagattagacgtaaggttcctttgctttcagaagaacttttaaatggaaaccctaatattgaaattccagatgacgaggaagcacatgtaaagaaggctttaaaaaaggaaaaagagggtcttccaaaggttttgaaaaaagagcacgatgaatttgttaaaaaaatgatggatgagtcagaaacaattaatattccagaggccttacaatatgacacaatgtatgaagatatactcagtaaaggagaaattcgaaaagaaatcaaaaaacaaatacctaatcctacaacatcttttgagagtatatcaatgacaactgaagaggaaaaagtcgacagtactttaaaaagcgaaatgcaaaatcgtgtctctaagccttcttttgatacctggtttaaaaacactaagatcaaaattgaaaataaaaattgtttattacttgtaccgagtgaatttgcatttgaatggattaagaaaagatatttagaaacaattaaaacagtccttgaagaagctggatatgttttcgaaaaaatcgaactaagaaaagtgcaataaactgctgaagtatttcagcagttttttttatttagaaatagtgaaaaaaatataatcagggaggtatcaatatttaatgagtactgatttaaatttatttagactggaattaataattaacacgtagactaattaaaatttaatgagggataaagaggatacaaaaatattaatttcaatccctattaaattttaacaagggggggattaaaatttaattagaggtttatccacaagaaaagaccctaataaaatttttactagggttataacactgattaatttcttaatgggggagggattaaaatttaatgacaaagaaaacaatcttttaagaaaagcttttaaaagataataataaaaagagctttgcgattaagcaaaactctttactttttcattgacattatcaaattcatcgatttcaaattgttgttgtatcataaagttaattctgttttgcacaaccttttcaggaatataaaacacatctgaggcttgttttataaactcagggtcgctaaagtcaatgtaacgtagcatatgatatggtatagcttccacccaagttagcctttctgcttcttctgaatgtttttcatatacttccatgggtatctctaaatgattttcctcatgtagcaaggtatgagcaaaaagtttatggaattgatagttcctctctttttcttcaacttttttatctaaaacaaacactttaacatctgagtcaatgtaagcataagatgtttttccagtcataatttcaatcccaaatcttttagacagaaattctggacgtaaatcttttggtgaaagaatttttttatgtagcaatatatccgatacagcaccttctaaaagcgttggtgaatagggcattttacctatctcctctcattttgtggaataaaaatagtcatattcgtccatctacctatcctattatcgaacagttgaactttttaatcaaggatcagtcctttttttcattattcttaaactgtgctcttaactttaacaactcgatttgtttttccagatctcgagggtaactagcctcgccgatcccgcaagaggcccggcagtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcatgc |
| pgrac100-MS | Bacillus subtilis-Escherichia coli shuttle plasmid with inserted SacB Promoter, RBS, pchB, pchA, BSMT1, and terminator; converts sucrose to methyl salicylate | ttaagttattggtatgactggttttaagcgcaaaaaaagttgctttttcgtacctattaatgtatcgttttagaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatattactgtaacataaatatatattttaaaaatatcccactttatccaattttcgtttgttgaactaatgggtgctttagttgaagaataaaagaccacattaaaaaatgtggtcttttgtgtttttttaaaggatttgagcgtagcgaaaaatccttttctttcttatcttgataataagggtaactattgccgatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcaggccttaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgttttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcatcaaaatcgtctccctccgtttgaatatttgattgatcgtaaccagatgaagcactctttccactatccctacagtgttatggcttgaacaatcacgaaacaataattggtacgtacgatctttcagccgactcaaacatcaaatcttacaaatgtagtctttgaaagtattacatatgtaagatttaaatgcaaccgttttttcggaaggaaatgatgacctcgtttccaccggaattagcttggtaccCCGCACATATACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATTAAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAAACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTATCAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTCGAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAGGGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGTAACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACATAAAAAAGGAGACATGAACGAAAGGAGGAAGGATCCATGATGAAAACACCGGAAGATTGCACAGGCCTGGCAGATATTAGAGAGGCGATTGATCGCATTGATCTGGATATTGTTCAGGCACTGGGCAGAAGAATGGATTATGTTAAAGCAGCATCACGCTTTAAAGCATCAGAAGCAGCAATTCCGGCACCGGAAAGAGTTGCAGCAATGCTGCCGGAAAGAGCAAGATGGGCAGAAGAAAATGGCCTGGATGCACCGTTTGTTGAAGGCCTGTTTGCACAGATCATCCACTGGTATATTGCAGAACAAATTAAATACTGGAGACAGACAAGAGGCGCAGCATGAGCAGACTGGCACCGCTGTCACAATGCCTGCATGCACTGAGAGGCACATTTGAAAGAGCAATTGGCCAAGCACAAGCACTGGATAGACCGGTTCTGGTTGCAGCAAGCTTCGAAATTGATCCGCTTGATCCGCTGCAAGTTTTTGGCGCATGGGATGATAGACAAACACCGTGCCTGTATTGGGAACAACCGGAACTGGCATTCTTTGCATGGGGCTGCGCACTGGAACTGCAAGGCCATGGCGAGCAACGCTTTGCACGTATTGAGGAAAATTGGCAACTGCTGTGCGCAGATGCAGTTGTTGAAGGCCCGCTGGCACCGAGACTGTGCGGAGGATTTAGATTTGATCCGAGAGGCCCGAGAGAAGAACATTGGCAAGCATTTGCAGATGCAAGCCTGATGCTGGCAGGCATTACAGTTCTGAGAGAAGGCGAAAGATATAGAGTTCTGTGCCAACATCTGGCAAAACCGGGCGAAGACGCACTGGCACTGGCAGCATATCATTGCAGCGCACTGCTGAGACTGAGACAACCGGCAAGAAGAAGACCGTCAGGCCCGACAGCAGGCGCACAAGGAGATGCATCAGCACAAGAAAGAAGACAATGGGAAGCAAAAGTTTCAGATGCAGTTTCATCAGTTAGACAAGGCAGATTTGGCAAAGTTGTTCTGGCAAGAACACAAGCAAGACCGCTGGGCGATATTGAACCGTGGCAAGTTATTGAACATCTGAGACTGCAACATGCAGATGCACAACTGTTTGCATGCAGAAGAGGCAATGCATGCTTTCTGGGCGCATCACCGGAAAGACTGGTTAGAATTAGAGCAGGCGAAGCACTGACACATGCACTGGCAGGCACAATTGCAAGAGGCGGCGATGCACAAGAAGATGCAAGACTGGGCCAAGCACTGCTGGATAGCGCAAAAGATAGACATGAACATCAACTGGTTGTTGAAGCAATTCGCACAGCACTGGAACCGTTTTCAGAAGTTCTGGAAATTCCGGATGCACCGGGCCTGAAAAGACTGGCAAGAGTTCAACATCTGAATACACCGATTAGAGCAAGACTGGCAGATGCAGGCGGCATTCTGAGACTGCTGCAAGCACTGCATCCGACACCGGCAGTTGGCGGCTATCCGAGATCAGCAGCACTGGATTATATTAGACAACATGAAGGCATGGATAGAGGCTGGTATGCAGCACCGCTGGGCTGGCTGGATGGCGAAGGAAATGGCGATTTTCTGGTTGCACTGAGATCAGCACTGCTGACACCGGGCAGAGGCTATCTGTTTGCAGGCTGCGGCCTGGTTGGAGATTCAGAACCGGCACATGAATATCGCGAAACATGCCTGAAACTGTCAGCAATGAGAGAAGCACTGTCAGCAATTGGCGGCCTGGATGAAGTTCCGCTGCAAAGAGGCGTTGCATAATCTAGAATGGAAGTTGTTGAAGTTCTGCATATGAATGGCGGCAATGGCGATTCAAGCTATGCAAATAACTCACTGGTTCAACAGAAGGTGATTCTGATGACAAAACCGATTACAGAACAAGCAATGATTGACCTGTATTCAAGCCTGTTTCCGGAAACACTGTGCATTGCAGATCTGGGCTGCTCACTGGGCGCAAATACATTTCTGGTTGTTTCACAACTGGTTAAAATCGTTGAGAAGGAGCGCAAAAAGCATGGCTTTAAAAGCCCGGAATTCTATTTCCATTTTAACGACCTGCCGGGCAATGATTTTAACACGCTGTTTCAATCACTGGGCGCATTTCAAGAGGACCTGAGAAAACATATTGGCGAATCATTTGGCCCGTGCTTTTTCTCAGGCGTTCCGGGCTCATTTTATACACGCCTGTTTCCGTCAAAGAGCCTGCATTTTGTTTATTCAAGCTATAGCCTGATGTGGCTGTCACAAGTGCCGAATGGCATTGAAAATAACAAAGGCAATATCTATATGGCACGCACAAGCCCGCTGAGCGTTATTAAAGCATACTACAAGCAGTACGAAATTGACTTCAGCAACTTCCTGAAGTACCGCTCAGAAGAACTGATGAAAGGCGGCAAAATGGTTCTGACACTGCTGGGCAGAGAATCAGAAGATCCGACAAGCAAAGAATGCTGCTATATTTGGGAACTGCTGGCAATGGCACTGAATAAGCTGGTTGAAGAGGGCCTGATTAAAGAAGAAAAAGTTGATGCATTTAACATCCCGCAGTATACGCCGAGCCCGGCAGAGGTTAAGTATATCGTCGAAAAGGAGGGCTCATTTACAATTAATCGCCTGGAAACATCAAGAGTTCATTGGAACGCATCAAACAATGAAAAGAACGGCGGCTATAATGTTAGCAGATGCATGAGAGCAGTTGCAGAACCGCTGCTGGTTTCACATTTTGATAAAGAACTGATGGATCTGGTCTTCCACAAGTACGAAGAGATTGTTTCAGATTGCATGAGCAAAGAAAACACGGAGTTCATTAACGTGATCATCTCACTGACGAAAATCAATTGACCCggggcagcccgcctaatgagcgggcttttttcacgtcacgcgtccatggagatctttgtctgcaactgaaaagtttataccttacctggaacaaatggttgaaacatacgaggctaatatcggcttattaggaatagtccctgtactaataaaatcaggtggatcagttgatcagtatattttggacgaagctcggaaagaatttggagatgacttgcttaattccacaattaaattaagggaaagaataaagcgatttgatgttcaaggaatcacggaagaagatactcatgataaagaagctctaaaactattcaataaccttacaatggaattgatcgaaagggtggaaggttaatggtacgaaaattaggggatctacctagaaagccacaaggcgataggtcaagcttaaagaacccttacatggatcttacagattctgaaagtaaagaaacaacagaggttaaacaaacagaaccaaaaagaaaaaaagcattgttgaaaacaatgaaagttgatgtttcaatccataataagattaaatcgctgcacgaaattctggcagcatccgaagggaattcatattacttagaggatactattgagagagctattgataagatggttgagacattacctgagagccaaaaaactttttatgaatatgaattaaaaaaaagaaccaacaaaggctgagacagactccaaacgagtctgtttttttaaaaaaaatattaggagcattgaatatatattagagaattaagaaagacatgggaataaaaatattttaaatccagtaaaaatatgataagattatttcagaatatgaagaactctgtttgtttttgatgaaaaaacaaacaaaaaaaatccacctaacggaatctcaatttaactaacagcggccaaactgagaagttaaatttgagaaggggaaaaggcggatttatacttgtatttaactatctccattttaacattttattaaaccccatacaagtgaaaatcctcttttacactgttcctttaggtgatcgcggagggacattatgagtgaagtaaacctaaaaggaaatacagatgaattagtgtattatcgacagcaaaccactggaaataaaatcgccaggaagagaatcaaaaaagggaaagaagaagtttattatgttgctgaaacggaagagaagatatggacagaagagcaaataaaaaacttttctttagacaaatttggtacgcatataccttacatagaaggtcattatacaatcttaaataattacttctttgatttttggggctattttttaggtgctgaaggaattgcgctctatgctcacctaactcgttatgcatacggcagcaaagacttttgctttcctagtctacaaacaatcgctaaaaaaatggacaagactcctgttacagttagaggctacttgaaactgcttgaaaggtacggttttatttggaaggtaaacgtccgtaataaaaccaaggataacacagaggaatccccgatttttaagattagacgtaaggttcctttgctttcagaagaacttttaaatggaaaccctaatattgaaattccagatgacgaggaagcacatgtaaagaaggctttaaaaaaggaaaaagagggtcttccaaaggttttgaaaaaagagcacgatgaatttgttaaaaaaatgatggatgagtcagaaacaattaatattccagaggccttacaatatgacacaatgtatgaagatatactcagtaaaggagaaattcgaaaagaaatcaaaaaacaaatacctaatcctacaacatcttttgagagtatatcaatgacaactgaagaggaaaaagtcgacagtactttaaaaagcgaaatgcaaaatcgtgtctctaagccttcttttgatacctggtttaaaaacactaagatcaaaattgaaaataaaaattgtttattacttgtaccgagtgaatttgcatttgaatggattaagaaaagatatttagaaacaattaaaacagtccttgaagaagctggatatgttttcgaaaaaatcgaactaagaaaagtgcaataaactgctgaagtatttcagcagttttttttatttagaaatagtgaaaaaaatataatcagggaggtatcaatatttaatgagtactgatttaaatttatttagactggaattaataattaacacgtagactaattaaaatttaatgagggataaagaggatacaaaaatattaatttcaatccctattaaattttaacaagggggggattaaaatttaattagaggtttatccacaagaaaagaccctaataaaatttttactagggttataacactgattaatttcttaatgggggagggattaaaatttaatgacaaagaaaacaatcttttaagaaaagcttttaaaagataataataaaaagagctttgcgattaagcaaaactctttactttttcattgacattatcaaattcatcgatttcaaattgttgttgtatcataaagttaattctgttttgcacaaccttttcaggaatataaaacacatctgaggcttgttttataaactcagggtcgctaaagtcaatgtaacgtagcatatgatatggtatagcttccacccaagttagcctttctgcttcttctgaatgtttttcatatacttccatgggtatctctaaatgattttcctcatgtagcaaggtatgagcaaaaagtttatggaattgatagttcctctctttttcttcaacttttttatctaaaacaaacactttaacatctgagtcaatgtaagcataagatgtttttccagtcataatttcaatcccaaatcttttagacagaaattctggacgtaaatcttttggtgaaagaatttttttatgtagcaatatatccgatacagcaccttctaaaagcgttggtgaatagggcattttacctatctcctctcattttgtggaataaaaatagtcatattcgtccatctacctatcctattatcgaacagttgaactttttaatcaaggatcagtcctttttttcattattcttaaactgtgctcttaactttaacaactcgatttgtttttccagatctcgagggtaactagcctcgccgatcccgcaagaggcccggcagtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcatgc |

## Polypeptide and Protein Sequences

In this module, you can find the polypeptide and amino acid sequences in the entire project. You can quickly locate the sequence you are looking for based on the name of the polypeptide or protein.

| Sequence Name | Function | Sequence (5'→3') |
| --- | --- | --- |
| **CP-Histag-CP** | A recombinant dimer expressing MS2 coat protein with His tag | ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYANFTQFVLVDNGGTHHHHHHGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY |
| **CP-GBP3.1-CP** | A recombinant dimer of MS2 coat protein with GBP3.1 localization peptide | ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYTCSKKYPRSPCMGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY |
| **CP-GBP3.1-CP-TAT-Histag** | A recombinant dimer of MS2 coat protein with GBP3.1 localization peptide, TAT cell-penetrating peptide, and His tag | ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYTCSKKYPRSPCMGDVTVAPSNFANGVAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIYQLQGGPAPGGSAYGRKKRRQRRRLEHHHHHH |
| EGFP | An enhanced green fluorescent protein used for tracing the function of GBP3.1 and TAT peptides | MVSFKSLLVLCCAALGAFATKRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK |
| GBP3.1-EGFP-TAT-Histag | Enhanced green fluorescent protein fused with GBP3.1 localization peptide, His tag, and TAT cell-penetrating peptide | TCSKKYPRSPCMGGGGSGGGGSGGGGSGGGGSMVSFKSLLVLCCAALGAFATKRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKANFKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKLQGGPAPGGSAYGRKKRRQRRRLQGGPAPGGSALEHHHHHH |
| TAT | Cell-penetrating peptide | YGRKKRRQRRR |
| GBP3.1 | Aphid gut localization peptide | TCSKKYPRSPCM |

## Conventional PCR Primer Sequences

In this module, you can find the primer sequences used in PCR throughout the entire project. You can quickly locate the primer you are looking for based on the name of the amplified gene or the species name.

| Amplified Gene or Sequence Name | Species | Primer Name | Primer Sequence (5'→3') | Product Length (bp) | Application |
| --- | --- | --- | --- | --- | --- |
| Triple fusion gene dsRNA & glufosinate resistance gene | ***Metarhizium anisopliae*** | pBar-3×dsRNA-F | gcccttcctccctttatttc | 711bp & 955bp | Used for screening successfully transformed *Metarhizium* after T-DNA insertion via Agrobacterium-mediated transformation |
|  |  | pBar-3×dsRNA-R | ctgacatcgacaccaacgat |  |  |
| SacB Promoter, RBS, pchB, pchA, BSMT1 genes | ***Bacillus subtilis*** | BS-F | GACCTCGTTTCCACCGGAAT |  |  |
|  |  | BS-R | TCTTCTTCCGTGATTCCTTG |  |  |
| **CP-Histag-CP** | Recombinant protein | CP F | CGTACGTGGTACCGGTATG | 353bp |  |
|  |  | CP R | AGCAGCCTAGGACATGGGTA |  |  |
| **CP-GBP3.1-CP-TAT-Histag** | Recombinant protein | CP F | CGTACGTGGTACCGGTATG | 353bp |  |
|  |  | CP R | AGCAGCCTAGGACATGGGTA |  |  |
| GBP3.1-EGFP-Histag-TAT | Recombinant protein | E2 F | CCACTACCAGCAGAACACTCC | 139bp |  |
|  |  | E2 R | AGCGGTCACGAACTCCAAC |  |  |
| EGFP | Recombinant protein | E4 F | AGTCGCTGCTGGTCTTATGC | 371bp |  |
|  |  | E4 R | GTAGTTGCCGTCGTCCTTGA |  |  |

## RT-PCR and qPCR Primer Sequences

In this module, you can find the RT-PCR and qPCR primer sequences used in the entire project. You can quickly locate the primer you are looking for based on the name of the amplified gene or the species name.

| Amplified Gene Name | Species | Primer Name | Primer Sequence (5'→3') | Product Length (bp) | Application |
| --- | --- | --- | --- | --- | --- |
| CP19 | *Toxoptera citricida* | *TCiCP*-3-F | TTCGATTACGCGGTTCACGA | 87 | qPCR for amplifying CP19 cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CP19 gene |
|  |  | *TCiCP*-3-R | GTACGCTCCCTTGACCACAT |
| *EF1α* | *Toxoptera citricida* | *TCiEF1α*-2-F | ACCTGCGAACATCACCACTG | 72 | qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CP19 gene |
|  |  | *TCiEF1α*-2-R | CCGGGAACAGCTTCTACCAA |
| *CHS* | *Toxoptera citricida* | *TCiCHS*-9-F | CAACGGCAAAAGACCCGAAC | 182 | qPCR for amplifying CHS cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CHS gene |
|  |  | *TCiCHS*-9-R | GCTTGCATGGATCGTCTCCT |
| *EF1α* | *Toxoptera citricida* | *TCiEF1α*-14-F | TGCTGTTGCTTTCGTTCCCA | 176 | qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CHS gene |
|  |  | *TCiEF1α*-14-R | CCTTGTCAGTTTGGCGACTG |
| *CYP450* | *Toxoptera citricida* | *TCiCYP*-14-F | ATCGACACGGCCACGTATTC | 121 | qPCR for amplifying CYP450 cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CYP450 gene |
|  |  | *TCiCYP*-14-R | CCGAAAACACGCACTCCTCT |
| *EF1α* | *Toxoptera citricida* | *TCiEF1α*-5-F | ACTGACAAGGCTCTCCGTCT | 149 | qPCR for amplifying EF1α cDNA from brown citrus aphid to evaluate the silencing effect of silencing RNA on the CYP450 gene |
|  |  | *TCiEF1α*-5-R | TCAACACGACCTACTGGGAC |
| Triple fusion gene dsRNA | ***Metarhizium anisopliae*** | pbar-jjz-F | GGCCGATCGAGATAAAGAACCA | 80 | qPCR for amplifying and evaluating dsRNA production efficiency in *Metarhizium* |
|  |  | pbar-jjz-R | TTTCTTCGTGGGTTTCGTGC |

Click to EXPAND the References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, *215*(3), 403–410. `https://doi.org/10.1016/S0022-2836(05)80360-2`

Asad, S., He, P., He, P., Li, Y., Wu, Y., Ahmed, A., Wang, Y., Munir, S., & He, Y. (2021). Interactions between Indigenous Endophyte ***Bacillus subtilis*** L1-21 and Nutrients inside Citrus in Reducing Huanglongbing Pathogen *Candidatus Liberibacter Asiaticus*. Pathogens, *10*(10), 1304. `https://doi.org/10.3390/pathogens10101304`

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{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
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SZU-China

2025
SZU-C
hina
2025

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# Results

"What I cannot create, I do not understand." . This page documents our creation, and therefore, embodies our
understanding.

![](https://static.igem.wiki/teams/5588/pagelogo/results.webp)

### Results

* [Overview](#section1)
* [Detection module](#section2)
  + [Background Information](#section2-2)
  + [Implementation Objectives](#section2-3)
  + [Verification process](#section2-4)
  + [Discussion](#section2-5)
* [RNA interference therapy](#section3)
  + [Background Information](#section3-2)
  + [Implementation Objectives](#section3-3)
  + [Verification process](#section3-4)
  + [Summary](#section3-5)
* [Delivery System](#section4)
  + [Background Information](#section4-2)
  + [Implementation Objectives](#section4-3)
  + [Verification process](#section4-4)
  + [Discussion](#section4-5)
* [Fungal Contact-Killing Module](#section5)
  + [Background Information](#section5-2)
  + [Implementation Objectives](#section5-3)
  + [Verification process](#section5-4)
  + [Discussion](#section5-5)
* [Overlook](#section6)
* [References](#section7)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Overview

**APHiGO** aims to combat aphid damage to citrus crops through safe and eco-friendly treatments,
addressing the
shortcomings
of existing chemical methods that result in environmental pollution. As described on the project webpage, the
application of chemical pesticides often leads to unavoidable chemical residues, jeopardizing the health and
balance of
organisms in the treated areas and even triggering the frightening consequences of chemical resistance. To
demonstrate
that **APHiGO** is a viable new approach for managing citrus aphid infestations, we have outlined
our validation
logic in
detail on the Experiment website. In this section, we will comprehensively present the key results from each
of the
validation modules.

### Detection Module

![Detection Module Results](https://static.igem.wiki/teams/5588/results/2-3.webp)

To monitor changes in aphid population density and thereby predict the optimal pesticide application
time, we constructed an engineered *Bacillus subtilis* strain capable of responding to sucrose—one
of the
main components of honeydew (aphid excrement). We designed a recombinant plasmid pgrac100-MS that can
convert sucrose into methyl salicylate (MeSA) and successfully transformed it into *Bacillus
subtilis*
168Δ4. After induction with sucrose, quantitative analysis via liquid chromatography-mass spectrometry
(LC-MS) showed that the MeSA yield reached 1.595 ng·mL⁻¹/OD under the induction concentration of 8%. This
engineered bacterium lays the foundation for both the strain and quantitative method required for the
early warning of citrus aphids.

[View Details →](#section2)

### RNA interference therapy

![RNA interference therapy Results](https://static.igem.wiki/teams/5588/results/3-25.webp)
![RNA interference therapy Results](https://static.igem.wiki/teams/5588/results/3-1.webp)

To screen for the RNA form with the highest insecticidal efficiency and its corresponding production
method, we carried out the optimization of RNA production conditions and analyzed the silencing and
lethal
effects. By adopting the bidirectional promoter and multi-terminator production strategy described by
Juliana da Rosa、SJ Ross et al., we successfully achieved the efficient preparation of RNA molecules. At
the same time, the fourth-generation RNA molecule we constructed using two natural miRNA scaffolds from
aphids, api-mir-71 and api-mir-3017a, exhibited a high aphid mortality rate of up to 60% after being
applied at a concentration of 800 ng/μl for 120 hours.

[View Details →](#section3)

### Delivery Module

![VLP Delivery System Results](https://static.igem.wiki/teams/5588/results/4-1.webp)

The susceptibility of RNA molecules to degradation during delivery is a major bottleneck restricting
their clinical application. To improve RNA stability, we developed a virus-like particle (VLP)-based
delivery system, specifically engineering MS2 VLPs capable of specifically packaging amiRNA via genetic
engineering approaches. MS2 VLPs are non-replicating nanoparticles based on the capsid protein of
bacteriophage MS2, exhibiting icosahedral symmetry. Herein, we successfully validated the integrity of the
capsid structure of this delivery system and its effective resistance to nucleases, and further explored
the feasibility of delivering it to the shoots of trees through trunk injection.

[View Details →](#section4)

### Fungal Contact-Killing Module

![Metarhizium Transformation Results](https://static.igem.wiki/teams/5588/home/process3.webp)

To enhance the virulence of *Metarhizium*, we modified *Metarhizium* via an
Agrobacterium-mediated genetic
transformation method to express target RNA molecules. This method is based on the ability of
*Agrobacterium* to transfer the T-DNA region on its Ti plasmid into host cells, thereby achieving
the
integration of the target gene. During the transformation process, *Agrobacterium* delivers T-DNA
into host
cells through its secretion system and integrates the target gene into the *Metarhizium* genome,
ensuring
the stable expression of the target gene in the fungus. Here, we successfully verified the ability of the
transformed *Metarhizium* to express the target RNA, and further analyzed the virulence of
*Metarhizium* at
the phenotypic level.

[View Details →](#section5)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Detection module

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Background Information

In March 2025, we conducted a research survey in Shanwei, Guangdong Province—a region with a high incidence
of citrus aphid infestations. It was found that citrus aphids are tiny in size and often hide on the
undersides of leaves, making them **difficult to detect**. This frequently causes farmers to miss
the optimal pesticide application time. Based on this observation, the team aimed to develop a biosensor
component capable of **responding to the population density of citrus aphids**.

Aphids excrete honeydew immediately after feeding; the amount of honeydew is positively correlated with
their population density, and one of its main components is **sucrose** Using this
characteristic, we designed and constructed a biosensor that can respond to sucrose signals. Taking Bacillus
subtilis—a biocontrol bacterium approved as a biopesticide—as the chassis, we implanted the
following genetic components: ① a sucrose-sensing element; ② the pchBA gene cluster (which can convert
chorismate, a metabolic product of sucrose, into salicylic acid (SA));③BSMT methyltransferase (which can
convert salicylic acid into **volatile MeSA**) Furthermore, the methyl salicylate
produced by this engineered bacterium can be recognized by **hardware devices**, enabling
real-time **monitoring of pest density**. This fundamentally solves the problem of aphids being
"difficult to detect" and advances the vision of precision agriculture and ecological sustainability.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Implementation Objectives

1. Construct an engineered *Bacillus subtilis* strain capable of expressing MeSA under sucrose
induction.

2.Conduct a preliminary evaluation to determine whether the engineered strain can respond to
changes in sucrose concentration, thereby expressing methyl salicylate at different concentrations.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Verification process

### Plasmid construction

pgrac100 is a commonly used *E. coli*-*Bacillus subtilis* shuttle vector, containing two
resistance
genes (for
ampicillin and chloramphenicol). When conducting protein expression in *Bacillus subtilis*,
chloramphenicol can
be used for screening.

We inserted the following genetic elements into the Multiple Cloning Site (MCS): ①
**sacB promoter**, a sucrose-inducible promoter; ② Ribosome Binding Site (RBS); ③
***pchA*** expression cassette: expresses isochorismate synthase, which catalyzes the
conversion of
chorismate to isochorismate; ④ *pchB* expression cassette: expresses isochorismate pyruvate lyase,
which
converts isochorismate into pyruvate and salicylate; ⑤ ***BSMT1*** expression cassette:
methylates
salicylate to produce methyl salicylate (a volatile ester); ⑥ terminator.

![pgrac100-MS recombinant plasmid](https://static.igem.wiki/teams/5588/results/2-1.webp)

Fig 2-1.
Schematic diagram of the pgrac100-MS recombinant plasmid.

### Plasmid transformation

After constructing and amplifying the plasmid, we prepared competent cells of *Bacillus subtilis*
168Δ4 using
the medium-alternating culture method and transformed both the empty vector and the recombinant plasmid into
the cells. Single colonies clones were randomly picked from resistant plates, and after amplification, the
plasmids were extracted for PCR and double digestion.Plasmid PCR showed that the empty vector control yielded
a band at 1537 bp, while the recombinant plasmid showed the expected band at **1777 bp**. The
double digestion results each showed two bands corresponding to the plasmid vector and the digested fragment.
The empty vector digestion product yielded a band at 776 bp, while the recombinant plasmid digestion product
showed the expected band at **2968 bp**. These results confirmed the successful construction and
transformation of the recombinant plasmid.

![pgrac100-MS recombinant plasmid transformation results](https://static.igem.wiki/teams/5588/results/2-2.webp)

Fig 2-2.
Results of pgrac100-MS recombinant plasmid transformation
(a) Growth of colonies on chloramphenicol-resistant plates after plasmid transformation.
(b) Agarose gel electrophoresis results of plasmid PCR and restriction digestion. M: DL5000 Marker; 1: PCR
result of the empty vector, with the target band at 776 bp; 2: PCR result of the recombinant plasmid, with the
target band at 2968 bp; 3: digestion result of the empty vector, with the target band at 1537 bp; 4: digestion
result of the recombinant plasmid, with the target band at 1777 bp. The gel was prepared with 2% agarose and
run at 120 V for 45 minutes.

### Quantification of Methyl Salicylate by Liquid Chromatography-Mass Spectrometry

We induced the engineered *Bacillus subtilis* with sucrose at concentrations of 0%, 4%, and 8% (w/v)
for 18
hours, and quantified methyl salicylate (MeSA) using liquid chromatography-mass spectrometry (LC-MS). Based on
the standard curve, we confirmed after **diluting 5 times** the production of MeSA and determined
the yields to be **1 ng·mL⁻¹/OD, 1.379 ng·mL⁻¹/OD, and 1.595 ng·mL⁻¹/OD**, respectively.

![methyl salicylate expression levels](https://static.igem.wiki/teams/5588/results/2-3.webp)

Fig 2-3.
Expression levels of methyl salicylate
(a) Standard curve of methyl salicylate.(b) Methyl salicylate concentrations induced by different sucrose
concentrations (dilute 5 times and quantify). Since sucrose induction inhibits the growth of Bacillus subtilis
to varying degrees, the expression levels were normalized using OD values to exclude the influence of cell
number. The OD₆₀₀ values for induction at 0%, 4%, and 8% were 4.102, 3.263, and 2.194, respectively.

The PDF of the original file is provided below and can be viewed by clicking the link. The sample values
represent the content after a 5-fold dilution.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Discussion

In this part of the experiment, we successfully constructed and transformed the pgrac100-MS recombinant
plasmid, obtaining a genetically stable engineered *Bacillus subtilis* 168Δ4 strain. Through liquid
chromatography-mass spectrometry (LC-MS), we initially verified that the constructed **engineered strain
can express MeSA**.

However, in this experiment, the engineered *Bacillus subtilis* strain under 0%
sucrose induction concentration was detected to produce 1 ng·mL⁻¹/OD of methyl salicylate, indicating that
there is **basal leakage** in the system. Therefore, in subsequent experiments, we will test the
methyl salicylate expression level of the **empty vector** and use it as a control. To further
optimize this strain, we will attempt to introduce a more **stringent sucrose operon**, such as
the suc dual regulation system, to reduce leaky expression.

In addition, on the basis of the
qualitative verification in this module, we also initially confirmed that the engineered strain **can
respond to changes in sucrose concentration**, thereby expressing methyl salicylate at different
concentrations. In the future, we will establish a **strict sucrose response gradient**, and
combine it with the **aphid population density** and the detection limit of the detection
**hardware** to achieve a closed loop from qualitative response to quantitative early warning.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# RNA interference therapy

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Background Information

RNAi is an eukaryotic gene silencing mechanism that cuts RNA to form siRNA, assembles the RISC
complex, and degrades
target mRNA, leading to organism death. Due to its high specificity and environmental friendliness, RNAi has
become a
core technology for precise pest control in agriculture. It addresses key issues in citrus aphid management,
such as
pesticide resistance, environmental pollution, and poor stability of biological control methods. This module
focuses on
the entire RNAi process: screening essential aphid genes to ensure lethality; optimizing RNA design
(upgrading from
single-target dsRNA to dual/tri-fusion dsRNA, tri-shRNA, bi-amiRNA) to enhance efficiency and stability;
utilizing
bidirectional promoters and multiple terminators to achieve high-level dsRNA production and address
transcription
"read-through" issues; quantifying silencing efficiency via RT-qPCR and validating the effect of
fourth-generation RNA
through lethality tests. The goal is to optimize parameters and develop a practical control solution to
support the
green transformation of the citrus industry.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Implementation Objectives

1. Verify the quality of plasmid linearization products by electrophoresis, confirm that **multiple
T7
terminators** are more conducive to efficient dsRNA transcription than a single terminator, and
provide a reference for high-efficiency RNA production.

2. Validate the effectiveness of procedures such
as
plasmid transformation, double-enzyme digestion linearization, and in vitro transcription, and optimize the
transcription system to ensure the **stable synthesis** of target dsRNA.

3. Clarify the
insecticidal activity of the **four generations of RNA** against aphids. Focus on verifying
**the synergistic insecticidal effect of fused dsRNA**, and evaluate the advantages of
tri-shRNA
and bi-amiRNA to support product iteration.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Verification process

### Optimization of a high-efficiency in vitro dsRNA production system

▼

The results showed that a transcription duration of 6 hours enabled relatively high yield in a short
time. By optimizing
the template with 3-tandem terminators and reverse-designed 3-tandem terminators, the phenomenon of
transcription
read-through was reduced. Compared with the template without terminators, the yield of dsRNA
synthesized via in vitro
transcription using the 3-tandem terminator template was increased.

![Figure 3-3](https://static.igem.wiki/teams/5588/results/3-4.webp)

Fig
3-3. (a) Significance analysis of transcription product concentrations for NT, 1T, 2T, and 3T
templates at 1-fold dilution; (b) significance analysis of transcription product concentration for
NT, 1T, 2T, and 3T templates at 5-fold dilution; (c) significance analysis of transcription product
concentration for NT, 1T, 2T, and 3T templates at 10-fold dilution; (d) linear regression curves
showing the relationship between dilution ratio and concentration.

If you are interested, please click here to learn more about our detailed experimental procedures.

To investigate the impact of different terminators on RNA production efficiency, we constructed dsRNA
expression vectors containing a **single T7 terminator and multiple T7 terminators** for
in vitro
transcription. Moreover, we set restriction enzyme sites between each terminator to enable **the
conversion between templates with different terminators**. We observed the band patterns of
the
products via gel electrophoresis, analyzed the yield and purity of the products, and determined the
effectiveness of the terminators.
We first prepared linear templates of NT, 1T, 2T, and 3T (xT
represents
x terminators, and NT represents no terminator). Through agarose gel verification, we successfully
**obtained the linear templates in the target band region**.

![Figure 3-1](https://static.igem.wiki/teams/5588/results/3-2.webp)

Fig
3-1. (a)MW:marker1,2:TAKARA DL1000 DNA marker; marker3: BBI DNA Marker A
(25~500 bp);1:*CHS*-1T(456bp);2:*CHS*-2T(594bp).(b)MW:marker(left):TAKARA DL1000 DNA
marker;
marker(right): BBI DNA Marker A (25~500 bp) ;1:*CHS*-NT(346bp);2:*CHS*-3T(716bp);xT
stands for x
terminators, and NT stands for no terminator.

We performed in vitro transcription on different templates separately. In Fig 3-2. (a) ,the
transcription product of the
**3T template showed relatively higher purity** compared with the others, but the
difference in
yield among them was not obvious in Fig 3-2. (b).

![Figure3-2](https://static.igem.wiki/teams/5588/results/3-3.webp)

Fig
3-2. (a) is a schematic diagram of agarose gel electrophoresis analysis for
purity comparison; (b) is a schematic diagram of agarose gel electrophoresis analysis for yield
comparison. MW: Marker; NT-*CHS*: *CHS* double-stranded RNA transcribed from the
terminator-free template;
1T-*CHS*: *CHS* double-stranded RNA transcribed from the template with the T7UUCG
terminator;
2T-*CHS*: *CHS* double-stranded RNA transcribed from the template with two tandem
terminators (T7UUCG
terminator and rrnBT1 terminator);
3T-*CHS*: *CHS* double-stranded RNA transcribed from the template with three tandem
terminators (T7UUCG
terminator, rrnBT1 terminator, and T7 terminator).

To more accurately determine the transcription
product
yield of templates with different terminators, we diluted the RNA samples 1-fold, 5-fold, and 10-fold
with
nuclease-free water respectively, then measured their concentrations using a Nanodrop. The test was
repeated 3
times, with nuclease-free water serving as the blank group. We calculated the average value of the
obtained
concentration data, established linear regression equations between the dilution factor and the
diluted
concentration, and then calculated the original concentration and conducted significance analysis. The
linear
regression equations for NT, 1T, 2T, and 3T are as
follows:YNT=6317\*X-29.37,Y1T=9538\*X+164.8,
Y2T=9604\*X-100.3,Y3T=11006\*X+240.3.

where X represents the dilution ratio (C now/C original) and is
dimensionless, and Y represents the concentration corresponding to the dilution ratio, with the unit
of ng/μL.
Fig 3-3, we found that the concentration of dsCHS transcribed using the template with
**tandem triple terminators (3T) was significantly higher** than that of templates with
other
terminators.

![Figure3-3](https://static.igem.wiki/teams/5588/results/3-4.webp)

Fig
3-3. (a) Significance analysis of transcription product concentrations for NT,
1T, 2T, and 3T templates at 1-fold dilution; (b) significance analysis of transcription product
concentration for NT, 1T, 2T, and 3T templates at 5-fold dilution; (c) significance analysis of
transcription product concentration for NT, 1T, 2T, and 3T templates at 10-fold dilution; (d) linear
regression curves showing the relationship between dilution ratio and concentration.
NT-*CHS*：YNT=6317\*X-29.37,R2=0.9967；1T-*CHS*：Y1T=9538\*X+164.8,R2=0.9997；2T-*CHS*：
Y2T=9604\*X-100.3,R2=0.9969；3T-*CHS*：Y3T=11006\*X+240.3,R2=0.9971。ns：p>0.05,\*：0.05,\*\*：p
<0.01,\*\*\*：p<0.001,\*\*\*\*：p<0.0001< /p>

We also investigated the effect of transcription duration on transcription efficiency.
We
transcribed the same batch of plasmid templates for 4 h, 6 h, and 12 h respectively, measured the
concentration of the products after different dilutions, and performed significance analysis on the
results
(as shown in Fig 3-4).It was found that the concentration of the transcription products after 6 h and
12 h
was significantly higher than that after 4 h, but there was no significant difference between 6 h and
12 h.
Therefore, after comprehensive consideration, **6 h was determined to be the more efficient
transcription duration**.

![Figure 3-4](https://static.igem.wiki/teams/5588/results/3-5.webp)

Fig
3-4. (a) Significance analysis of product concentrations after 4 h, 6 h, and
12 h of transcription at 2-fold dilution; (b) significance analysis of product concentrations at
5-fold dilution. ns: p > 0.05, \*: p < 0.05, \*\*: p < 0.01.

The results showed that a **transcription duration of 6
hours
enabled relatively high yield in a short time**. By optimizing the template with 3-tandem
terminators
and reverse-designed 3-tandem terminators, **the phenomenon of transcription read-through was
reduced**. Compared with the template without terminators, the **yield** of
dsRNA
synthesized via in vitro transcription using the 3-tandem terminator template **was
increased**.

### Single-target dsRNA Stage

▼

For the verification of ds*CYP450* and ds*CP*, nuclease-free water was used as the
control, and aphid
nymphs were treated
with the corresponding dsRNA solutions at 800 ng/μl. The aphid pretreatment, preparation of feeding
devices, and culture
conditions were all consistent with those of the *CHS* dsRNA experiment, ensuring the efficiency
of dsRNA
absorption and
the comparability of data. Results calculated using the same mortality correction formula showed that at
120 hours, the
corrected mortality rate of aphids in the 800 ng/μl ds*CP* treatment group reached 30%, which was
significantly higher
than the 20.48% of ds*CHS* at the same concentration; the corrected mortality rate of the
ds*CYP450*
treatment group further
increased to 31.4%, showing the strongest lethal activity among the three single-target dsRNAs.

![Figure3-10](https://static.igem.wiki/teams/5588/results/3-11.webp)

Fig
3-10. (a): Cumulative mortality rates of *Toxoptera citricida* (brown citrus
aphids) treated with 400 ng/μl dsCHS and 800 ng/μl dsCHS, compared with the control group, at 6 h, 12
h, 24 h, 36 h, 48 h, 60 h, 72h, 84h, 96h, 108h and 120 h post-treatment. Figure (b): Cumulative
mortality rates of *Toxoptera citricida* (brown citrus aphids) treated with 800 ng/μl dsCHS,
800 ng/μl
dsCYP450, and 800 ng/μl dsCP, compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h,
72h, 84h, 96h, 108h and 120 h post-treatment.

If you are interested, please click here to learn more about our detailed experimental procedures.

#### Preliminary Preparation

To ensure the smooth progress of subsequent RT-qPCR experiments, we conducted a preliminary
experiment in
advance to verify the primers of genes involved in qPCR. However, since the genes of the species
selected in
our experiment have not been fully annotated, we could not ensure the specificity of the designed
primers
through exon-spanning design or genomic BLAST during primer screening. Instead, we could only design
primers
based on the cDNA templates of the corresponding genes, and our options were limited. Therefore, we
synthesized a large number of primers for further screening.Following the advice of Expert Bai
Yongsheng, the
length of the internal reference fragment amplified by the primers used for qPCR should be similar to
that of
the target gene fragment to ensure consistent amplification efficiency, thereby reducing experimental
errors.
He suggested that we could use cDNA reverse-transcribed from the RNA of *Toxoptera citricida*
(brown
citrus
aphid) as a template for conventional PCR, and **preliminarily determine the specificity of the
primers** based on the results of 3.5% agarose gel electrophoresis to conduct the preliminary
screening of primers.

![Fig 3-5](https://static.igem.wiki/teams/5588/results/3-6.webp)

**Fig 3-5.** Figure (a) shows the agarose gel electrophoresis results of conventional PCR
for the positive reactions of primers CHS1–8 and their negative control (NC); Figure (b) shows the
electrophoresis results for the positive reactions of primers sets CHS9–12 and CP1–4, along with their
negative control (NC); Figure (c) shows the results for the positive reactions of primer sets CP5–7
and CYP450 1–5, along with their negative control (NC); and Figure (d) shows the results for the
positive reactions of primers sets CYP450 8–13 and their negative control (NC).

![Fig 3-6](https://static.igem.wiki/teams/5588/results/3-7.webp)

**Fig 3-6.** Figure (e) shows the agarose gel electrophoresis results of conventional PCR
for the positive reactions of primers EF1α1–8 and their negative control (NC); Figure (f) shows the
electrophoresis results for the positive reactions of primers EF1α9–16 and their negative control
(NC); Figure (g) shows the supplementary electrophoresis results for abnormal lane results observed
during the primer validation process.

From the preliminary screening results of conventional PCR and the
corresponding
negative control results, we observed that some primers exhibited non-specific amplification of short
fragments. Additionally, the appearance of multiple fragments in some negative control reactions
indicated
potential contamination of the existing primers. Therefore, we discarded the non-specific primers and
replaced
the contaminated ones based on the electrophoresis results. For the primers that passed the
preliminary
screening, we **further characterized their specificity** using qPCR melting curve
analysis.
After the characterization, the primers we finally selected are as follows:

![Fig 3-7](https://static.igem.wiki/teams/5588/results/3-8.webp)

Fig
3-7. Figure of qPCR primer screening results

#### Design

Through **literature review and screening in the NCBI database**, we identified a set of
potential targets for the brown citrus aphid. Using the **BLAST** tool to analyze the
homology of
these target genes, we set a homology threshold of 30% and retained **genes candidates**
below
this threshold to minimize off-target risks. We then selected closely related species (aphid pests)
with
sequence homology above 85% for alignment **analysis in MEGA**, identifying
**highly
conserved regions** of the gene within aphid pests to pinpoint efficient siRNA target sites.
Next, we
screened **high-scoring siRNA** fragments from multiple **public databases**
and,
based on these, prioritized dsRNA fragments that maximally covered the previously selected
high-scoring siRNA
sequences, **ultimately generating dsRNA molecules 300–500 bp in length**.

Through
these
screening steps, we obtained a 300 bp dsCHS, a 329 bp dsCYP450, and a 363 bp dsCP spanning the
full-length
sequence. All resulting sequences were validated by **BLAST** to ensure high specificity
and
controllable off-target risk.

#### Build

##### Plasmid Amplification

We performed transformation and amplification of the constructed plasmid template
**pUC57\_CHS-tri-terminator** by introducing it into competent DH5α cells (an *E.
coli*
strain
suitable for efficient routine plasmid cloning). The colonies grew well, and distinct single colonies
were
visible. We then expanded the bacterial culture to amplify the plasmid, obtaining a large quantity of
the
plasmid template.

##### Preparation of Linear Template

We verified the obtained plasmid template by double-enzyme digestion and agarose gel electrophoresis
to
ensure that we had the correct plasmid containing the DNA template (target band at 722 bp). After
confirming
the correctness of the plasmid, we performed double-enzyme digestion to prepare the linear template
for
transcription.

##### Transcription Reaction

We carried out bidirectional promoter-mediated in vitro transcription on the above-obtained linear
template
to obtain the target gene dsRNA. After transcription, we verified the product by agarose gel
electrophoresis
and successfully detected a dsRNA band at 352 bp, with minimal signs of degradation visible on the
gel. We
also prepared dsCYP450 and dsCP using the same workflow.

![Figure3-8](https://static.igem.wiki/teams/5588/results/3-9.webp)

Figure3-8: Agarose gel electrophoresis verification of dsCHS transcription products,dsCHS: 300
bp,Marker (left): TAKARA DL500 DNA Marker,Marker (right): TAKARA DL1000 DNA Marker

#### Test

##### Lethal Efficiency Verification of Single-target dsRNA

To clarify the lethal effects of the dsRNAs targeting *CHS*, *CP*, and *CYP450*
that we designed and
produced, and
to identify the optimal **concentration for single-target dsRNA, we first conducted a
concentration
gradient test on dsCHS**. Using nuclease-free water as the blank control, we prepared dsCHS
solutions
at concentrations of 400 ng/μl and 800 ng/μl. For each group, 30 aphid nymphs in consistent
physiological
condition were selected (nine groups in total), and all were subjected to a 3-hour fasting
pretreatment. The
aphids were then transferred to feeding devices containing the corresponding dsCHS solution or
nuclease-free
water; the devices were preloaded with citrus shoots sterilized with 75% ethanol, allowing the aphids
to
acquire and absorb dsRNA by feeding on the shoots.

All feeding devices were placed in an artificial
climate
chamber with conditions set to 25 ± 1°C, 70 ± 5% relative humidity, and a 16:8-hour photoperiod
(light:dark).
The number of dead aphids was observed and recorded every 12 hours, and fresh feeding devices were
replaced
every two days (to ensure continuous dsRNA supply and maintain normal aphid survival). The final
mortality was
counted after 120 hours.

![Fig 3-9](https://static.igem.wiki/teams/5588/results/3-10.webp)

Fig
3-9. Schematic diagram of the feeding device

The results showed that calculated using the mortality correction
formula: at
120 hours, the corrected mortality rate of aphids in the 400 ng/μl dsCHS treatment group was only
11.22%; in
contrast, the corrected mortality rate of the 800 ng/μl dsCHS treatment group increased significantly to
20.48%
at the same time point (Fig 3-10. (a)). This result not only confirms that the *CHS* dsRNA we
designed and
produced
has a clear lethal effect on aphids, but also indicates that its lethal effect is
concentration-dependent—**with the 800 ng/μl concentration showing better efficacy. Therefore, we
uniformly adopted the 800 ng/μl dsRNA concentration in the subsequent verification experiments for
dsCYP450
and dsCP**.

The corrected mortality rate formula is as follows:

![Corrected mortality rate formula](https://static.igem.wiki/teams/5588/results/6614904f-fc17-4926-92fa-52099b3e594c.webp)

For the verification of dsCYP450 and dsCP, nuclease-free water was used as the control, and aphid nymphs
were
treated with the corresponding dsRNA solutions at 800 ng/μl. The aphid pretreatment, preparation of
feeding
devices, and culture conditions were all consistent with those of the *CHS* dsRNA experiment,
ensuring the
efficiency of dsRNA absorption and the comparability of data. Results calculated using the same
mortality
correction formula showed that at 120 hours, the corrected mortality rate of aphids in the 800 ng/μl
dsCP
treatment group reached **30%**, which was significantly higher than the
**20.48%** of
dsCHS at the same concentration; the corrected mortality rate of the dsCYP450 treatment group further
increased
to **31.4%**, showing the strongest lethal activity among the three single-target dsRNAs
(Fig 3-10. (b)).

![Figure3-10](https://static.igem.wiki/teams/5588/results/3-11.webp)

Fig
3-10. (a): Cumulative mortality rates of *Toxoptera citricida* (brown citrus aphids) treated
with
400 ng/μl dsCHS and 800 ng/μl dsCHS, compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h,
60 h,72h,84h,96h,108h and 120 h post-treatment. Figure (b): Cumulative mortality rates of
*Toxoptera
citricida* (brown citrus aphids) treated with 800 ng/μl dsCHS, 800 ng/μl dsCYP450, and 800 ng/μl
dsCP,
compared with the control group, at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h,72h,84h,96h,108h and 120 h
post-treatment.

##### Silencing Efficiency Verification of Single-target dsRNA

To quantitatively assess the silencing efficiency of single-target dsRNAs on their target genes, we
collected
aphid samples treated with 800 ng/μl dsCHS, dsCP, dsCYP450, and the corresponding nuclease-free water
control
for 120 hours after completing the lethality experiment, and analyzed the expression levels of the
target
genes through the following experiments.

**1.Extraction and Quality Testing**

We extracted total RNA from aphids in each treatment group using the phenol–chloroform method. After
extraction, we precisely determined the RNA concentration and purity using a NanoDrop
spectrophotometer.
Meanwhile, RNA integrity was assessed by 1% agarose gel electrophoresis. The results showed that the
28S rRNA
and 18S rRNA bands were clear, sharp, and bright, with **the brightness of the 28S rRNA band
approximately twice that of the 18S rRNA band**. This indicated that the extracted RNA had
excellenstrong integrity, with minimal degradation or contamination, and fully met the requirements
for
subsequent experiments.

![Figure3-11](https://static.igem.wiki/teams/5588/results/3-12.webp)

Figure 3-11: Results of total RNA integrity detection in aphid samples by 1% agarose gel
electrophoresis. MW represents the molecular weight marker (100–5000 bp); Lanes 1–4 correspond to
total RNA from different aphid samples.

**2.Reverse transcription to synthesize cDNA**

From the above-qualified RNA samples, we synthesized cDNA via reverse transcription using the
TransScript®
One-Step gDNA Removal and cDNA Synthesis SuperMix kit. The specific reaction system and conditions
referred to
the protocol.

**3.Silencing efficiency measurement**

Using EF1α as the internal reference gene, we performed qPCR analysis using the SYBR Green method.
Each cDNA
sample was analyzed with three technical replicates, and the reaction mixture was prepared in a
96-well plate.
Using the nuclease-free water treatment group as the calibrator sample, we calculated the relative
gene
expression levels using the ΔΔCt method, and then derived the silencing efficiency.

The results
showed that
the CHS gene silencing rate in the dsCHS treatment group was **27%**, the CP gene
silencing rate
in the dsCP treatment group was **43%**, and the CYP450 gene silencing rate in the
dsCYP450
treatment group was 30% (Fig 3-12) These results indicate that the designed **single-target
dsRNAs can
effectively silence the corresponding target genes and show significant differences compared with
the
control group**.

![Figre 3-12](https://static.igem.wiki/teams/5588/results/3-13-2.webp)

Fig
3-12. The relative expression levels of *CHS*, *CYP450*, and *CP19*(*CP*)
in *Toxoptera citricida* after treatment
with 800 ng/μl dsCHS, dsCYP450, and dsCP for 120 h, respectively, compared to the control. Error bars
represent the standard deviation (SD) derived from at least three biological replicates. All datasets
were statistically analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P <
0.0001).

### Fused multi-target dsRNA

▼

The results showed that after 5 days, the corrected mortality rate of aphids in the dsF2 treatment
group reached 40.8%,
which was significantly higher than that of the single-target dsRNA (maximum 31.4%); the corrected
mortality rate in the
dsF3 treatment group further increased to 44.9%, which was not only superior to the single-target dsRNA
but also higher
than the dual-fusion dsRNA (Fig 3-15. (a)). These findings fully confirm the synergistic effect of the
dual-target RNAi
strategy and further highlight the stronger insecticidal advantage of triple-target synergy.

![Figure 3-15](https://static.igem.wiki/teams/5588/results/3-16-2.webp)

Figure 3-15.(a): Cumulative mortality rates of brown citrus aphids treated with 800 ng/μl dual-fusion
dsRNA (dsF2) or triple-fusion dsRNA (dsF3) at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108
h, and 120 h, compared with the control. Figure (b): Relative expression levels of CHS and CYP450 in
brown citrus aphids treated with 800 ng/μl dsF2 for 120 h, compared with the control.Error bars
represent the standard deviation (SD) derived from at least three biological replicates. All datasets
were statistically analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P <
0.0001). Figure (c): Relative expression levels of CHS, CYP450, and CP19(CP) in brown citrus aphids
treated with 800 ng/μl dsF3 for 120 h, compared with the control.Error bars represent the standard
deviation (SD) derived from at least three biological replicates. All datasets were statistically
analyzed using a Student's t-test (\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001).

If you are interested, please click here to learn more about our detailed experimental procedures.

#### Design

In the first-generation design, we successfully constructed dsRNA targeting a single key gene of the
brown
citrus aphid. After discussions with Dr. Chen Ruoyu, we decided to construct a multi-target fusion dsRNA
for
the second generation, aiming to enhance the lethal effect by targeting multiple key genes of the aphid.
Based
on the results of the first-generation experiments, we selected **short sequences (60–100 bp) with
high
specificity and high conservation**, and fused these optimized short fragments in a
**direct
tandem** arrangement. **RNAfold prediction analysis** was used to avoid
introducing
complex structures or repeat sequences at the gene fusion sites.
Ultimately, we obtained a dual-target fusion dsRNA (dsF2, targeting CHS and CYP450) and a triple-target
fusion
dsRNA (dsF3, targeting CHS, CYP450, and CP). **The dsRNAEngineer tool was used to ensure no
off-target
risk**.

#### Build

##### Preparation of Linear Template

We transformed the plasmids **pUC57\_dsF2 and pUC57\_dsF3**, which contain the target dsDNA
templates, into competent DH5α cells for plasmid amplification, yielding large quantities of the plasmid
templates harboring the target dsDNA. Double-enzyme digestion followed by agarose gel electrophoresis
showed clear, correct bands without any non-specific bands, confirming that we had successfully
prepared
high-quality DNA templates suitable for subsequent transcription.

##### Transcription Reaction

We used the resulting linear templates for in vitro transcription, followed by agarose gel
electrophoresis
verification, which confirmed that we had obtained dsRNA products **dsF2 and dsF3** of the
expected length.

![Figure 3-13](https://static.igem.wiki/teams/5588/results/3-14.webp)

Fig
3-13. Agarose gel electrophoresis verification
of dsF2 and dsF3 transcription products.(a)dsF2:402bp，marker(left):sangon 1000bp
marker，marker(right):BBI DNA Marker A (25~500 bp) (b)dsF3：391bp，marker:sangon 1000bp marker

#### Test

##### Lethal Efficiency Verification of Fused multi-target dsRNA

To investigate the synergistic lethal effect of fused dsRNA, we used nuclease-free water as the control
and,
based on the optimal concentration of 800 ng/μl determined in the single-target dsRNA experiments,
treated
aphid nymphs with solutions of **dual-fusion dsF2 and triple-fusion dsF3**, respectively.
Each
group contained 30 aphids, with a total of nine groups. After a 3-hour fasting pretreatment, the aphids
were
transferred to the corresponding feeding devices. The feeding devices and culture conditions were
identical
to
those in the single-target dsRNA experiments. During the experiment, aphid survival and mortality were
recorded every 12 hours, and fresh devices were replaced every two days, with continuous observation for
5
days (120 h). During the experiment, it was observed that some aphids turned red all over upon death,
and
some
died during molting.

![Figure 3-14](https://static.igem.wiki/teams/5588/proofofconcept/img-v3-02qs-08453295-692d-41eb-a1af-1eb82d37bc7g.webp)

Fig
3-14. (a) Observed under a stereomicroscope at
40× magnification, after brown citrus aphids were treated with 800 ng/μl triple-fusion multi-target
RNA (dsFA3), the individual on the left is a dead one with a whole-body reddening, and the individual
on the right is a normal one that is black; (b) Observed under a stereomicroscope at 40×
magnification, an individual of brown citrus aphid that died during the molting process after being
treated with 800 ng/μl triple-fusion multi-target RNA (dsFA3).

The results showed that after 5 days, the corrected mortality rate of aphids in the
**dsF2** treatment group reached **40.8%**, which was significantly higher
than
that
of the single-target dsRNA (maximum 31.4%); the corrected mortality rate in the **dsF3**
treatment group further increased to **44.9%**, which was not only superior to the
single-target
dsRNA but also higher than the dual-fusion dsRNA (Fig 3-15. (a)). These findings fully confirm the
synergistic
effect of the dual-target RNAi strategy and further highlight the stronger insecticidal advantage of
triple-target synergy.

##### Silencing Efficiency Verification of Fused multi-target dsRNA

Following the same workflow of RNA extraction by the phenol–chloroform method, cDNA synthesis, and
qRT–PCR
detection, we verified the silencing effects of dual-fusion and triple-fusion dsRNA. **The results
showed that** after **dsF2** treatment, the silencing rate of the CYP450 gene was
31.5%
(Fig 3-15. (b)); after dsF3 treatment, the silencing rates of the CHS, CYP450, and CP genes were
**35.3%,
20%, and 45%**, respectively(Fig 3-15. (c)). These results further demonstrate the role of
fused
multi-target dsRNA in gene silencing and provide a molecular basis for its synergistic insecticidal
effect.

![Figure 3-15](https://static.igem.wiki/teams/5588/results/3-16-2.webp)

Fig
3-15. (a): Cumulative mortality rates of brown
citrus aphids treated with 800 ng/μl dual-fusion dsRNA (dsF2) or triple-fusion dsRNA (dsF3) at 6 h, 12
h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, compared with the control. Figure (b):
Relative expression levels of CHS and CYP450 in brown citrus aphids treated with 800 ng/μl dsF2 for
120 h, compared with the control. Error bars represent the standard deviation (SD) derived from at
least three biological replicates. All datasets were statistically analyzed using a Student's t-test
(\*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001). Figure (c): Relative expression levels of CHS,
CYP450, and CP19(CP) in brown citrus aphids treated with 800 ng/μl dsF3 for 120 h, compared with the
control. Error bars represent the standard deviation (SD) derived from at least three biological
replicates. All datasets were statistically analyzed using a Student's t-test (\*P < 0.05; \*\*P <
0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001).

### Multitandem short hairpin RNA stage

▼

The results showed that after 120 h (5 days), the corrected mortality rate of aphids in the tri-shRNA
treatment group
reached 49.5% (Fig 3-18.), demonstrating effective killing of aphids and showing better insecticidal
efficacy compared
with the triple-fusion dsRNA (44.9%), thus providing a technical basis for subsequent RNAi product
iteration.

![Figure 3-18](https://static.igem.wiki/teams/5588/results/3-19.webp)

Figure 3-18.Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72
h, 84 h, 96 h, 108 h, and 120 h after treatment with 800 ng/μl tri-shRNA and in the control group.

If you are interested, please click here to learn more about our detailed experimental procedures.

#### Design

Due to the susceptibility of dsRNA to degradation and its relatively high off-target risk, we developed
the
third-generation RNA molecule design—shRNA—based on the multi-target approach. **shRNA improves
RNA
stability and reduces off-target effects through more precise sequence design**. We used the
TTCAAGAGA universal hairpin sequence and linked multiple shRNA hairpin units with a linker, successfully
designing a tri-shRNA targeting CHS, CP, and CYP450. We performed RNA secondary structure simulations
using
RNAfold to confirm the structural independence of each shRNA unit and analyzed off-target effects using
dsRNAEngineer, ultimately obtaining tri-shRNA.

![Figure 3-17](https://static.igem.wiki/teams/5588/results/3-17.webp)

Figure 3-16.tri-shrna二级结构预测Secondary structure prediction of tri-shRNA

#### Build

##### Plasmid transformation

We transformed the plasmid containing the target dsDNA template into competent cells—DH5α, an *E.
coli*
strain
suitable for efficient plasmid transformation—for subsequent plasmid amplification.

##### Plasmid Amplification

We inoculated the transformed *E. coli* via the streak plate method to obtain single colonies.
Subsequently,
we
expanded the number of bacteria by culturing the obtained single colonies in a shaking flask, thereby
obtaining more plasmids containing the target template for subsequent template preparation.

##### Preparation of Linear Template

We used restriction endonucleases to act on the restriction site located after the target sequence on
the
plasmid, obtaining restriction digestion products for the subsequent transcription system. The
single-enzyme
digestion gap plays a role as a physical terminator in the subsequent transcription experiment, which is
also
used for the subsequent transcription system. The gel electrophoresis results showed that we obtained a
single
restriction digestion product with the correct length.

##### Transcription Reaction

We added the obtained linear template to the system of the transcription kit, and obtained tri-shRNA
products
with the expected length through transcription.

![Figure 3-18](https://static.igem.wiki/teams/5588/results/3-18-2.webp)

Fig
3-17. Marker ( left ) : DL5000 DNA Marker 1 : negative control 2、3、4 : tri-shRNA transcription
product——213bp Marker ( right ): D2000 DNA Marker

#### Test

##### Lethal Efficiency Verification of Multitandem short hairpin RNA

Using nuclease-free water as the control, we treated aphid nymphs with a multitandem short hairpin RNA
(tri-shRNA) solution at a concentration of 800 ng/μl. The feeding devices and culture conditions were
the
same
as in the single-target dsRNA experiments, and we continuously observed and recorded aphid survival and
mortality. The results showed that after 120 h (5 days), the corrected mortality rate of aphids in the
tri-shRNA treatment group reached **49.5%** (Fig 3-18) demonstrating effective killing of
aphids
and showing better insecticidal efficacy compared with the triple-fusion dsRNA (44.9%), thus providing a
technical basis for subsequent RNAi product iteration.

![Figure 3-19](https://static.igem.wiki/teams/5588/results/3-19.webp)

Fig
3-18. Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84
h, 96 h, 108 h, and 120 h after treatment with 800 ng/μl tri-shRNA and in the control group.

### bi-Artificial miRNA stage

▼

The results showed that after 120 h (5 days), the corrected mortality rate of aphids in the bi-amiRNA
treatment group
reached as high as 60.2% (Fig 3-22.), demonstrating the optimal insecticidal efficacy among the four
generations of RNA
molecules.

![Figure 3-22](https://static.igem.wiki/teams/5588/results/3-23.webp)

Fig
3-22. Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72
h, 84 h, 96 h, 108 h, and 120 h after treatment with 800 ng/μL bi-amiRNA and in the control group
(CTRL).

If you are interested, please click here to learn more about our detailed experimental procedures.

#### Design

We aimed to **more effectively utilize endogenous RNAi-related proteins in organisms**,
and
based on this, we designed the fourth-generation RNA molecule—multitandem artificial miRNA (amiRNA).
Compared
with the above three generations of RNA molecules (which induce RNAi through the exogenous siRNA
pathway),
this fourth-generation molecule uses the natural miRNA precursor scaffold of organisms, which
**can
trigger the endogenous microRNA processing mechanism in eukaryotes to further improve RNAi
efficiency**. We screened the miRBase database and obtained two natural miRNA scaffolds from
aphids—**api-mir-71 and api-mir-3017a**. The affinity of the scaffolds ensures the
processing
efficiency of the RNA molecules.

![Figure 3-20](https://static.igem.wiki/teams/5588/results/3-20.webp)

Figure 3-19.Skeletal structures of api-mir-71 (a) and api-mir-3017a (b), where the pink parts
represent the final mature miRNA sequences.

We integrated the previously screened **CYP450** siRNA
sequence into **api-mir-71** and the **CHS** siRNA sequence into
api-mir-3017a,
then
linked them using a universal linker (AGGCAT) to obtain our artificial miRNA (amiRNA). Similarly, we
performed
RNA secondary structure simulation via RNAfold to ensure the independence of each hairpin unit.

![Figure 3-21](https://static.igem.wiki/teams/5588/results/img-v3-02qr-0d736604-39b9-4082-8a47-2e21cc60013g.webp)

Figure 3-20. Prediction of the secondary structure of bi-amiRNA

#### Build

##### Plasmid transformation

We transformed the plasmid containing the target dsDNA template into competent cells—DH5α, an E. coli
strain
suitable for efficient plasmid transformation—for subsequent plasmid amplification.

##### Plasmid Amplification

We inoculated the transformed E. coli using the streak plate method to obtain single colonies.
Subsequently,
we increased the bacterial count by culturing the obtained single colonies in a shaking flask, thereby
acquiring more plasmids containing the target template for subsequent template preparation.

##### Preparation of Linear Template

We used restriction endonucleases to act on the restriction site located downstream of the target
sequence
on
the plasmid, obtaining restriction digestion products for the subsequent transcription system. The
single-enzyme digestion gap functions as a physical terminator in the subsequent transcription
experiment
and
is also used in the subsequent transcription system. Gel electrophoresis results showed that we obtained
a
single restriction digestion product with the correct length.

##### Transcription Reaction

We added the obtained linear template to the system of the transcription kit, and obtained bi-amiRNA
products
with the expected length through transcription.

![Figure 3-22](https://static.igem.wiki/teams/5588/results/bmi.avif)

Figure 3-21. Agarose gel electrophoresis analysis of bi-amiRNA transcription products.Marker ( left )
: DL5000 DNA Marker；1 : negative control；2、3、4 : bi-amiRNA transcription product——157bp；Marker ( right
) : D2000 DNA Marker

#### Test

##### Lethal Efficiency Verification of Artificial miRNA (amiRNA)

Using nuclease-free water as the control, we treated young aphids with an artificial miRNA (amiRNA)
solution
at a concentration of 800 ng/μL. The feeding devices and culture conditions were consistent with those
in
the
dsRNA experiment, and we continuously observed and recorded the survival and mortality of the aphids.
The
results showed that after 120 h (5 days), the corrected mortality rate of aphids in the bi-amiRNA
treatment
group reached as high as **60.2%** (Fig 3-22), demonstrating the optimal insecticidal
efficacy
among the four generations of RNA molecules.

![Figure 3-23](https://static.igem.wiki/teams/5588/results/3-23.webp)

Figure 3-22.Cumulative mortality rates of brown citrus aphids at 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72
h, 84 h, 96 h, 108 h, and 120 h after treatment with 800 ng/μL bi-amiRNA and in the control group
(CTRL).

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Summary

In this study, by optimizing the high-efficiency in vitro dsRNA production system, it was found that the
use
of **multi-terminator** templates significantly increased the yield of dsRNA, and 6 hours was
determined as the optimal transcription duration. Furthermore, **four generations of RNA
molecules** were designed and their insecticidal effects on citrus aphids were verified. The data
in
the table below clearly shows the mortality rate and silencing efficiency of each generation of RNA
molecules.
Among them, the fourth-generation artificial miRNA (bi-amiRNA) exhibited the highest aphid mortality rate
after 120 hours, reaching 60.2%. These results provide an important basis for the development of efficient
and
specific RNAi-based control strategies against citrus aphids.

Table:
Summary Table of RNAi Therapeutic Outcomes

![Figure 3-24](https://static.igem.wiki/teams/5588/results/3-24.webp)

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Delivery System

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Background Information

Virus-like particles (VLPs) are hollow protein nanoparticles self-assembled from one or more structural
proteins of viruses. They are highly similar to natural viruses in size, shape, and surface structure, but the
most fundamental difference is that they **completely lack the viral genetic material (DNA or
RNA)**; thus, they possess no replication or infection capabilities and **exhibit high
biosafety**.

The core advantage of VLPs stems from their unique property of being "virus-like but
not viruses": the highly ordered and repetitively arranged antigenic epitopes on their surface can be
efficiently recognized by the immune system, thereby triggering strong humoral and cellular immune
responses—making them an ideal platform for vaccine development. Currently, vaccines based on VLP technology,
such as human papillomavirus (HPV) vaccines and hepatitis B vaccines, have been successfully marketed and
widely used worldwide. Beyond prophylactic vaccines, VLPs also show great potential as multifunctional
nanocarriers. **Their internal cavity can load and protect nucleic acid drugs (mRNA, siRNA,
shRNA)**, and they can achieve cell- or tissue-targeted delivery through engineering modifications
like surface display technology. Therefore, they hold broad prospects in fields such as targeted drug therapy
and gene editing. Among them, VLPs derived from MS2 viruses can specifically bind to nucleic acids with pac
sequences and rapidly assemble into a polymer composed of 180 capsid proteins (CP).

This year, **the
SZU-China team successfully constructed a VLP vector platform using MS2 phage capsid proteins. Through
surface display technology, they co-displayed the TAT transmembrane peptide (which enhances cell
penetration) and the GBP3.1 peptide (which specifically targets aphid intestinal cells) on the particle
surface, thereby endowing this VLP system with the function of cross-species targeted delivery**. In
this section, we will present the characterization of the engineered VLPs and their stress resistance.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Implementation Objectives

We aim to construct a virus-like particle (VLP)-based RNA delivery system to address the key challenge of RNA
being easily degraded during delivery to aphids. We will prepare VLPs capable of efficiently packaging target
RNA through genetic engineering methods and focus on evaluating their capsid integrity and resistance to
exogenous nucleases, thereby providing experimental evidence for the development of a stable and efficient RNA
delivery platform.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Verification process

### Plasmid construction and Characterization of Protein

▼

The results showed that the eluate containing the target protein exhibited a distinct bright band at
the predicted
position, which is consistent with our prediction that the His-tagged VLPs have a molecular weight of
approximately 29
kDa. This preliminarily confirms that we have successfully obtained the His-tagged VLP proteins.

![Fig 4-5. SDS-PAGE analysis](https://static.igem.wiki/teams/5588/results/4-6.webp)

Fig
4-5. SDS-PAGE analysis of the purified target protein expressed by pACYCDuet\_1-CP-Histag-CP-amiRNA
stained with Coomassie Brilliant Blue. Lane MW is the protein molecular weight marker; Lane 1 is the
bacterial culture supernatant, Lane 2 is the flow-through during purification; Lanes 3 and 4 are the
washing solutions in the purification process of the target protein, and Lanes 5–9 are the purified
eluates. The theoretical molecular weight of the target protein is 28333.00 Da (approximately 28 kDa),
and the actual size of the electrophoretic band is about 28 kDa, which is consistent with the
theoretical molecular weight.

If you are interested, please click here to learn more about our detailed experimental procedures.

### Plasmid construction

### 1.Construction of plasmid pACYCDuet\_1-CP-Histag-CP-amiRNA

The construction of plasmid pACYCDuet\_1-CP-Histag-CP-amiRNA is shown in Fig 4-1. In this construction,
the MS2 capsid
protein dimer–6×His tag fusion gene (CP–6×His–CP) was placed downstream of a T7 promoter.
Simultaneously, two RNA
interference sequence fragments targeting aphid genes—namely, chitin synthase (api-mir-71) and CYP450
(api-mir-3017a)—were cloned into specific sites of the plasmid. A 4,640 bp recombinant plasmid was
successfully
constructed. This plasmid can express the MS2 capsid protein and transcribe RNA carrying target gene
sequences—specifically, a bifunctional amiRNA capable of silencing both the chitin synthase gene
(api-mir-71) and the
cytochrome CYP450 gene.

![Figure 4-1. Schematic diagram of plasmid](https://static.igem.wiki/teams/5588/results/4-2.webp)

Fig
4-1. Schematic diagram of plasmid pACYCDuet\_1-CP-Histag-CP-amiRNA.

### 2.Construction of plasmid pET28a\_CP-GBP3.1-CP-TAT-Histag

The construction of plasmid pET28a\_CP-GBP3.1-CP-TAT-Histag aimed to obtain MS2 capsid proteins
displaying functional
peptides on their surface. As shown in Fig 4-2, we constructed plasmid pET28a\_CP-GBP3.1-CP-TAT-Histag
(6076 bp). In this
plasmid, a fusion gene sequence—MS2 capsid protein (CP)–aphid intestinal targeting peptide
(GBP3.1)–second MS2 capsid
protein (CP)—was obtained via gene synthesis. The C-terminus of this sequence was linked to the TAT
cell-penetrating
peptide via a flexible linker peptide, and a 6×His purification tag was added immediately after the TAT
to facilitate
affinity chromatography purification. This fusion gene was cloned downstream of the T7 promoter.
Therefore, upon
induction, the plasmid can express a single-chain version of the capsid protein dimer, whose surface can
functionally
display both the GBP3.1 targeting peptide and the TAT cell-penetrating peptide.

![Fig 4-3. Schematic diagram of plasmid](https://static.igem.wiki/teams/5588/results/4-3.webp)

Fig
4-2. Schematic diagram of plasmid pET28a\_CP-GBP3.1-CP-TAT-Histag.

### 3.Construction of plasmid pET28a\_GBP3.1-eGFP-TAT-HisTag

To obtain a vector capable of expressing a multifunctional fusion protein incorporating the aphid
intestinal
targeting peptide (GBP3.1), the enhanced green fluorescent protein (eGFP) reporter gene, and the HIV-1
trans-membrane peptide (TAT), we constructed plasmid **pET28a\_GBP3.1-eGFP-TAT-HisTag**(6214 bp).
As shown in Figure X, the complete fusion gene
sequence—GBP3.1-linker-eGFP-linker-TAT-linker-6×HisTag—was
obtained via gene synthesis and cloned into the multiple cloning site (MCS) of the pET-28a(+) vector,
placing
its expression under the control of the T7/lac hybrid promoter. Under IPTG-induced conditions, this
plasmid
can be expressed at high efficiency in an E. coli host that expresses T7 RNA polymerase (e.g.,
BL21(DE3)),
producing a fusion protein with a C-terminal 6×His tag for subsequent purification. The inclusion of
eGFP
provides a visual tracking tool for protein expression localization and the purification process.

![Fig 4-4. Schematic diagram of plasmid](https://static.igem.wiki/teams/5588/results/4-4.webp)

Fig
4-3. Schematic diagram of plasmid pET28a\_CP-GBP3.1-CP-TAT-Histag.

### 4.Construction of plasmid pET28a\_eGFP

As a control, we constructed plasmid pET28a\_eGFP (6085 bp). As shown in Figure 2, the eGFP gene
sequence was
cloned into the multiple cloning site (MCS) of the pET-28a(+) vector, placing its expression under the
control
of the T7/lac hybrid promoter. This plasmid is used to express the enhanced green fluorescent protein
alone in
the corresponding host strain.

![Fig 4-5. Schematic diagram of plasmid](https://static.igem.wiki/teams/5588/results/4-5.webp)

Fig
4-4. Schematic diagram of plasmid pET28a\_eGFP.

### Characterization of the single-chain version of the capsid protein dimer

After constructing the above plasmids, these recombinant plasmids were transformed into Escherichia
coli BL21 (DE3), and
positive monoclonal colonies were selected on antibiotic-resistant plates for expansion culture and
colony PCR. Our
specific primers successfully amplified the corresponding target bands, confirming the successful
transformation of the
plasmids.
After inducing protein expression with IPTG, the His-tagged virus-like particle (VLP) proteins were
purified by nickel
affinity chromatography. The results after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are shown
in (Fig 4-5. and
Fig 4-6.). The results showed that the eluate containing the target protein exhibited a distinct bright
band at the
predicted position, which is consistent with our prediction that the His-tagged VLPs have a molecular
weight of
approximately 29 kDa. This preliminarily confirms that we have successfully obtained the
His-tagged VLP proteins.

![Fig 4-5. SDS-PAGE analysis](https://static.igem.wiki/teams/5588/results/4-6.webp)

Fig
4-5. SDS-PAGE analysis of the purified target protein expressed by pACYCDuet\_1-CP-Histag-CP-amiRNA
stained with Coomassie Brilliant Blue. Lane MW is the protein molecular weight marker; Lane 1 is the
bacterial culture supernatant, Lane 2 is the flow-through during purification; Lanes 3 and 4 are the
washing solutions in the purification process of the target protein, and Lanes 5–9 are the purified
eluates. The theoretical molecular weight of the target protein is 28333.00 Da (approximately 28 kDa),
and the actual size of the electrophoretic band is about 28 kDa, which is consistent with the
theoretical molecular weight.

![Fig4-6. SDS-PAGE analysis](https://static.igem.wiki/teams/5588/results/4-7.webp)

Fig
4-6. SDS-PAGE analysis of the purified target protein expressed by pET28a\_CP-GBP3.1-CP-TAT-Histag,
stained with Coomassie Brilliant Blue. Lane MW represents the protein molecular weight marker; Lane 1
is the bacterial culture supernatant, Lane 2 is the flow-through during the purification process;
Lanes 3 and 4 are the bacterial pellets, and Lanes 5–9 are the purified eluates. The theoretical
molecular weight of the target protein is 31042.25 Da (approximately 31 kDa), while the actual size of
the electrophoretic band is about 30 kDa, which is consistent with the theoretical molecular weight.

### Characterization of MS2 VLPs

▼

In this experiment, we characterized the assembly of MS2 VLPs particles. Through TEM detection,
individual particles showed uniform spherical structures with clear boundaries. These particle
morphologies demonstrate that amiRNA and proteins can effectively assemble into VLPs nanoparticles.

![TEM images of MS2 VLPs](https://static.igem.wiki/teams/5588/results/4-9.webp)

Fig
4-8. Transmission Electron Microscopy (TEM) images of purified histidine-tagged MS2 phage-like
particles (histidine-tagged VLPs). The scale bar represents 100 nanometers.

If you are interested, please click here to learn more about our detailed experimental procedures.

### 1.Characterization of VLPs

To verify whether histidine-tagged virus-like particles (VLPs) successfully encapsulate the target RNA
inside their
capsids, we designed and conducted a combined verification experiment. First, the samples were treated
with nucleases
for gradient time periods; subsequently, agarose gel electrophoresis was used to analyze and record the
retention of the
target RNA. After the nucleic acid detection was completed, the same gel was stained with Coomassie
Brilliant Blue R250
to detect the presence of VLP capsid proteins. The experimental results showed that the target RNA could
still be stably
detected after nuclease treatment, and the capsid protein staining result was positive(Fig 4-7.). This
confirms that the
target RNA was successfully encapsulated inside the protein capsids of the VLPs.

![Agarose gel characterization of VLPs](https://static.igem.wiki/teams/5588/results/4-8.webp)

Fig
4-7. Agarose gel images for characterization of the assembled products. We first stained with Super
Red nucleic acid dye to detect RNA (Panel A), followed by Coomassie Brilliant Blue R-250 staining to
detect MS2 coat protein (Panel B). Lane MW (the leftmost lane in both Panels A and B) shows the DNA
ladder. In Panel A, Lanes 1–4 contain 50 µM virus-like particles (VLPs) samples treated with 100 U/mL
Benzonase nuclease at 37 °C for 90 min, 60 min, 30 min, and 0 min, respectively; in Panel B, Lanes 5–8
correspond to Lanes 1–4 in Panel A. The results show that Lanes 1–4 corresponding to VLPs exhibit a
bright RNA band above 1000 bp; due to the large overall size of RNA encapsulated within the capsid
proteins, its migration speed in the gel is slower. It can be observed that the shorter the nuclease
treatment time, the more distinct the band becomes; after nuclease treatment, VLP aggregates
depolymerizes and releases amiRNA, thus a bright amiRNA band can be observed in the 100–200 bp range.
It can be observed that the shorter the nuclease treatment time, the brighter the band; notably, under
the condition of 0 min nuclease treatment, RNA is encapsulated within the protein capsid, making it
difficult to observe discrete RNA bands. A comparison of the lanes patterns in Panels A and B confirms
that the target RNA is successfully encapsulated inside the protein capsids of the VLPs.

### 2.Transmission electron microscopy (TEM) experiments

To verify whether amiRNA and capsid proteins can assemble into virus-like particles (VLPs) from a
physical morphological
perspective, we performed negative-staining transmission electron microscopy (TEM) analysis on the
purified samples.
Samples with a protein concentration of 50 μM (determined by the Bradford method) were dropped onto a
hydrophilically
treated ultra-thin support film copper grid, stained negatively with phosphotungstic acid (PTA), and
observed under an
accelerating voltage of 80 kV. The TEM results are shown in Fig 4-8.: in the low-magnification field of
view in Fig 4-8.
A, large-scale aggregates formed by capsid proteins can be observed, and this morphology is comparable
to the research
results of LaNell A. Williams et al. (2024); the high-magnification field of view in Fig 4-8. B clearly
shows typical
VLP clusters, in which individual particles exhibit a uniform spherical structure with clear boundaries.
The morphology
of these particles confirms that amiRNA and proteins can effectively assemble into VLP nanoparticles.

![TEM images of MS2 VLPs](https://static.igem.wiki/teams/5588/results/4-9.webp)

Fig4-8. Transmission Electron Microscopy
(TEM) images of purified histidine-tagged MS2 phage-like particles (histidine-tagged VLPs). The scale
bar represents 100 nanometers.

### Verification of MS2 VLPs Function

▼

The results showed that, after treatment with Benzonase Nuclease at 37°C for 10 min, the naked RNA was
completely
degraded; under the same conditions, the RNA encapsulated within MS2 capsid proteins remained
significantly protected,
indicating that VLP particles exhibit remarkable stability against nucleases.

![Nuclease resistance of VLPs](https://static.igem.wiki/teams/5588/results/4-10.webp)

Fig
4-9. Agarose gel image showing the nuclease resistance of the assembled products.The gel was stained
with Super Red nucleic acid dye to detect amiRNA. Lanes MW (leftmost and rightmost lanes) contain a
DNA ladder (molecular weight marker). Lanes 1–4 contain 50 µM virus-like particles (VLPs) treated with
100 U/mL Benzonase nuclease at 37 °C for 0 min, 10 min, 20 min, and 30 min, respectively. Lanes 6–9
contain 500 nM amiRNA treated under the same conditions as the VLPs (100 U/mL Benzonase nuclease, 37
°C) for 0 min, 10 min, 20 min, and 30 min, respectively. Lane 5 is a blank control, and Lane 10 is a
control containing capsid protein (CP) dimers without amiRNA.The results show that Lanes 1–4,
corresponding to VLPs, exhibit a bright RNA band above 1000 bp. Because the RNA encapsulated within
the capsid proteins has a large overall size, its migration speed in the gel is slower. It can be
observed that the shorter the nuclease treatment time, the more distinct the band becomes. After
nuclease treatment, VLP aggregates depolymerize and release amiRNA, thus a bright amiRNA band can be
observed in the 100–200 bp range. Again, the shorter the nuclease treatment time, the brighter this
band appears. Notably, under the condition of 0 min nuclease treatment, the RNA is encapsulated within
the protein capsid, making it difficult to observe discrete RNA bands. Furthermore, the RNA content in
Lane 1 (VLPs treated for 0 min) is higher than that in Lane 6 (amiRNA treated for 0 min). This is
presumably because RNA is prone to degradation in the natural environment, and the amiRNA degraded
rapidly during loading and electrophoresis. A comparison of the two groups of lanes reveals that RNA
encapsulated in VLPs still exhibits a better protective effect in a high-nuclease environment.

If you are interested, please click here to learn more about our detailed experimental procedures.

### Investigation of the Protective Effect of VLP Nanoparticles on amiRNA

To evaluate the protective effect of VLP nanoparticles on amiRNA, we performed a time-gradient
experiment using
Benzonase Nuclease. The experimental setup was as follows: the control group contained 750 ng of naked
RNA, and the
experimental group contained 50 µM His-tagged VLPs. Both groups were treated with 100 U/mL Benzonase
Nuclease at 37°C
for 0 min, 10 min, 20 min, and 30 min. The remaining RNA content was then detected by agarose gel
electrophoresis (Fig
4-9.).The results showed that, after treatment with Benzonase
Nuclease at
37°C for 10 min, **the naked RNA was completely degraded**; under the same conditions,
**the RNA encapsulated within MS2 capsid proteins remained significantly protected, indicating
that VLP
particles exhibit remarkable stability against nucleases**.

![Nuclease resistance of VLPs](https://static.igem.wiki/teams/5588/results/4-10.webp)

Fig
4-9. Agarose gel image showing the nuclease resistance of the assembled products.The gel was stained
with Super Red nucleic acid dye to detect amiRNA. Lanes MW (leftmost and rightmost lanes) contain a
DNA ladder (molecular weight marker). Lanes 1–4 contain 50 µM virus-like particles (VLPs) treated with
100 U/mL Benzonase nuclease at 37 °C for 0 min, 10 min, 20 min, and 30 min, respectively. Lanes 6–9
contain 500 nM amiRNA treated under the same conditions as the VLPs (100 U/mL Benzonase nuclease, 37
°C) for 0 min, 10 min, 20 min, and 30 min, respectively. Lane 5 is a blank control, and Lane 10 is a
control containing capsid protein (CP) dimers without amiRNA.The results show that Lanes 1–4,
corresponding to VLPs, exhibit a bright RNA band above 1000 bp. Because the RNA encapsulated within
the capsid proteins has a large overall size, its migration speed in the gel is slower. It can be
observed that the shorter the nuclease treatment time, the more distinct the band becomes. After
nuclease treatment, VLP aggregates depolymerize and release amiRNA, thus a bright amiRNA band can be
observed in the 100–200 bp range. Again, the shorter the nuclease treatment time, the brighter this
band appears. Notably, under the condition of 0 min nuclease treatment, the RNA is encapsulated within
the protein capsid, making it difficult to observe discrete RNA bands. Furthermore, the RNA content in
Lane 1 (VLPs treated for 0 min) is higher than that in Lane 6 (amiRNA treated for 0 min). This is
presumably because RNA is prone to degradation in the natural environment, and the amiRNA degraded
rapidly during loading and electrophoresis. A comparison of the two groups of lanes reveals that RNA
encapsulated in VLPs still exhibits a better protective effect in a high-nuclease environment.

### Validation of Delivery Function

Previous studies have shown that MS2-encapsulated nucleic acid molecules exhibit poor ability to escape
from
endosomes after cellular phagocytosis (Reference). Furthermore, unmodified VLP-RNAi complexes distribute
randomly within aphids, making it difficult for most small RNA molecules to be efficiently taken up and
exert
their effects. To enhance the uptake and delivery efficiency of VLPs in the aphid gut (Reference), we
systematically fused two functional peptides to the surface of the MS2 capsid protein: **GBP3.1**(a 12-amino-acid intestinal binding peptide: TCSKKYPRSPCM) and **TAT** (an
11-amino-acid
cell-penetrating peptide: YGRKKRRQRRR).

We transformed the constructed plasmid into *Escherichia coli*
BL21 (DE3) and expanded the culture. Following induction
with IPTg, the His-tagged CP-GBP3.1-TAT fusion protein was purified using nickel affinity
chromatography. SDS-PAGE
electrophoresis results showed that the His-tagged CP-GBP3.1-TAT fusion protein was successfully
expressed and purified
(Fig 4-6.).

### Western Blot Detection of Tree Tissues Following Vascular Injection of VLPs

To verify whether virus-like particles (VLPs) can be efficiently transported to leaves and stems via
vascular tissue
injection in trees, we designed an experimental system using red ink as a tracer molecule and VLPs as
the functional
carrier, referencing the principles of trunk injection technology. Purified VLP proteins were mixed with
red ink at a
specific ratio (for the preparation method, see Table 4-1.). A continuous injection device (Fig 4-10.)
was used to
perform targeted injection into the vascular tissue of citrus trees for three days. This device can
provide a constant
propelling force for the agent and record the injection dosage.

After the injection, young stem and leaf tissues were collected from the trees, and total protein was
extracted for
Western Blot (WB) analysis (Fig 4-11). The results showed that clear target protein
bands
were detected in leaf samples from all sampling sites. This indicates that **VLPs can be
transported to
the apical young tissues along with the tree's transpiration stream, and the injection device achieved
controlled and sustained delivery**.

Table
4-1 Injection material

![Image 4-11-2](https://static.igem.wiki/teams/5588/results/4-11-2.webp)

![Image 4-12](https://static.igem.wiki/teams/5588/results/4-12.webp)

Fig
4-10. Schematic diagram of the injection device

![Figure 4-11](https://static.igem.wiki/teams/5588/results/4-13.webp)

Fig
4-11. Analysis of VLP distribution in the young stems of citrus trees after injection with the mixed
agent of red ink and VLPs via Western Blot. MW represents the protein molecular weight marker; NC is
the negative control of untreated young stems; Lane Stem-injection is the collected protein sample
from young stems. The results show that a bright band is visible at approximately 28 kDa in the
experimental group sample, while there is no corresponding band in the NC lane. This indicates that
VLPs can be effectively delivered via vascular injection and distributed in young stem tissues.

To verify the tissue targeting and cell penetration
efficiency of GBP3.1 and TAT transmembrane peptides in aphids, we designed a fusion protein expression
system
using enhanced Green Fluorescent Protein (EGFP) as the reporter gene. By constructing prokaryotic
expression
vectors of the pET series, we expressed the GBP3.1-EGFP-TAT fusion protein, which was then purified via
His-tag affinity chromatography. The purified fusion protein was used to treat aphids through a feeding
method, with a separate EGFP group set as the negative control.Observations were conducted respectively
via in
vivo imaging using a fluorescence microscope, and via observation of tissue sections subjected to
immunofluorescent staining with an enhanced green fluorescent protein (EGFP) antibody using a laser
confocal
microscope.

The results showed that, compared with the control group, the TAT-EGFP and GBP3.1-EGFP
treatment groups **exhibited significantly enhanced retention of green fluorescent signals in
aphids.
This indicates that both TAT and GBP3.1 can effectively carry EGFP to penetrate the tissue barriers of
aphids and enter the interior of cells, with GBP3.1 showing a certain degree of tissue targeting
specificity**. These results visually confirm the effectiveness and feasibility of GBP3.1 and
TAT as
delivery carriers in living aphids, and provide key evidence for the effectiveness of functional
peptides for
the subsequent construction of a delivery system that combines MS2 virus-like particles (MS2 VLPs)
loaded with
target RNA and the GBP3.1 and TAT transmembrane peptides. For more information and results regarding the
delivery system, you can visit the [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proofofconcept%27%29%20%7D%7D) webpage to learn
more about this section in detail.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Discussion

This collection of studies provides solid theoretical and experimental evidence for the core
hypothesis—**“MS2 Virus-Like Particles (VLPs) modified with GBP3.1 (aphid midgut-targeting peptide) and
TAT (cell-penetrating peptide) enable efficient delivery of silencing small RNAs**”—through
systematic experiments spanning plasmid construction, VLP characterization, and functional validation.Based on
the results in this section and the results regarding the tracking-based validation of the functions of GBP3.1
and the TAT cell-penetrating peptide (CPP) on the proof of concept (PoC) wiki page, we believe that achieving
this objective is fully feasible.

The MS2 VLP system constructed in this study exhibits distinct
advantages. Compared to traditional delivery methods (e.g., direct dsRNA spraying or unmodified VLPs),
**this system more effectively protects nucleic acids from degradation and significantly enhances
cell-specific uptake efficiency due to the targeting capability of GBP3.1**. In addition, unlike
chemically synthesized polymeric nanocarriers, all components of this system possess excellent
biodegradability and biocompatibility. Notably, VLP technology platforms have been extensively explored for
drug delivery in humans, indicating that the system developed herein is an **environmentally and
human-friendly technology** with broad application potential across multiple fields.

Currently,
MS2 VLPs have been successfully self-assembled in *Escherichia coli* (*E. coli*). Transmission
Electron
Microscopy
(TEM) observations revealed that their morphology does not consist of uniformly dispersed individual spherical
particles; instead, they tend to aggregate in clusters. This finding is consistent with the report by LaNell
A. Williams et al. (2024), who identified the stoichiometric ratio of components in the assembly system as a
key factor determining VLP morphology. **We hypothesize that the complexity of the intracellular
environment in *E. coli* makes MS2 VLP difficult to precisely control this ratio, leading to the
formation of
capsid clusters**. This represents one of the limitations of using in vivo expression systems for MS2
VLP production.
Given this, **the dimers of recombinant MS2 VLPs may hold great potential for RNA packaging in
vitro**. It is also crucial to evaluate the RNA protection efficacy of both VLP capsid clusters and
dispersed morphologies. Importantly, the conformation of proteins fused to MS2 VLP dimers and their insertion
sites appear to exert a significant influence on assembly stability. The final morphology of MS2 VLPs strongly
depends on the conformation of the chimeric protein and its insertion site on the capsid, as well as the
density of the targeting peptide. In-depth investigation into the regulatory mechanisms of these factors on
VLP assembly will greatly expand the compatibility of MS2 VLPs with heterologous proteins. Notably, the use of
covalent ligation systems such as SpyTag/SpyCatcher can effectively circumvent assembly challenges caused by
the size or properties of fusion proteins, making this a highly promising solution.

In conclusion, the MS2
VLP system is poised to evolve into a modular RNA-based biopesticide delivery platform. Its application
prospects are not limited to aphid control; by simply replacing the targeting peptide (e.g., with specific
ligands targeting other pests or pathogens), the platform can be rapidly adapted to diverse biological
scenarios. **Therefore, establishing a modified modular platform for MS2 VLPs holds significant
potential, demonstrating remarkable versatility and flexibility**.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Fungal Contact-Killing Module

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Background Information

As a type of natural entomopathogenic fungus, *Metarhizium anisopliae* (commonly known as green
muscardine
fungus) can germinate after its spores come into contact with the cuticle of brown citrus aphids. The
germinated
spores then differentiate to form a specialized infection structure—appressoria—which further penetrates the
aphid’s cuticle, invades the hemolymph, and proliferates extensively. During this process, *M.
anisopliae*
secretes metabolites such as destruxins, which can disrupt the aphid’s tissue structure and ultimately lead to
the aphid's death.Therefore, *M. anisopliae* has been registered as an environment-friendly
biopesticide in many
countries worldwide, and is currently widely used in the agricultural production of various crops such as
citrus
and rice.

![Figure 5-1. Flowchart of Metarhizium Infecting Brown Citrus Aphids](https://static.igem.wiki/teams/5588/results/5-1.webp)

Fig 5-1.
Flowchart of *Metarhizium* Infecting Brown Citrus Aphids

However, the infection process of *Metarhizium* takes a certain amount of time, while brown citrus
aphids reproduce extremely rapidly—this makes traditional biopesticides unable to completely eliminate the
pests. In contrast, **using genetic engineering to modify fungal strains for expressing host-derived
small
RNAs can effectively overcome the drawback of slow infection speed**, achieving the desired pest
control effect, especially during the local outbreak and migration of aphids.

Therefore, the SZU-China team
designed dsRNAs targeting three genes of brown citrus aphids (the CHS gene, CYP450 gene, and CP19 gene),
inserted them into the T-DNA region of the Ti plasmid, and then introduced them into *Metarhizium* via
**Agrobacterium-mediated transformation. After transformation**, the target genes were integrated
into the *Metarhizium* genome, enabling the stable expression of dsRNAs in *Metarhizium
anisopliae* and
ensuring
their effective function.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Implementation Objectives

1. Using an Agrobacterium-mediated transformation method, the target fragment expressing the fusion dsRNA was
inserted into the genome of *Metarhizium anisopliae*, enabling its stable expression in
*Metarhizium*.

2. Clarify
the infection effect of *Metarhizium anisopliae* on brown citrus aphids.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Verification process

### Construction of plasmid pBar\_PtrpC-dsF3

For the validation of the component function, we chose to carry it in the T-DNA of the pBar vector (kindly
provided by
Professor Duo Lai from Shenyang Agricultural University). This region contains a tryptophan constitutive
promoter and a
glufosinate resistance gene, which are used to screen for successfully transformed strains.Furthermore, the
dsRNA we
designed was inserted downstream of the tryptophan constitutive promoter (PtrpC), and transcription
termination is
mediated by the downstream tryptophan terminator (ttrpC) (see Fig 5-2.). Ultimately, we constructed a plasmid
with a
length of 10,376 bp, in which the T-DNA fragment will be inserted into the genome of *Metarhizium* via
Agrobacterium-mediated transformation.This dsRNA is transcribed from an expression cassette consisting of a
forward DNA
sequence, a stem-loop, and a reverse DNA sequence, enabling simultaneous targeting of three genes in brown
citrus
aphids.

![Figure 5-2. Schematic representation of the pBar_PtrpC-dsF3 plasmid](https://static.igem.wiki/teams/5588/results/5-2.webp)

Fig 5-2.
Schematic diagram of the plasmid pBar\_PtrpC-dsF3-TtrpC.

### Agrobacterium-mediated genetic transformation

The plasmid was first transformed into *Agrobacterium*. Successfully transformed
*Agrobacterium* strains were
able to grow
normally on medium containing kanamycin, whereas strains transformed with the empty pBar vector could not
grow. Single
colonies from the resistant medium were picked for PCR verification, yielding amplified sequences of 711 bp
and 955 bp
in length (Fig 5-3. (a)), confirming that the transformed *Agrobacterium* carried the target plasmid.
Under induction with acetosyringone (AS) and MES, *Agrobacterium* transferred the Ti plasmid into
spores of
*Metarhizium
anisopliae*. After glufosinate resistance screening, successfully transformed *Metarhizium*
strains were able to
grow
normally on SDAY medium containing cefotaxime and glufosinate, whereas *Metarhizium* transformed with
the empty
pBar
vector could not grow (Fig 5-3. (b)).Twenty milligrams of mycelia from normally growing single-colony
*Metarhizium* were
used for genomic DNA extraction for PCR verification and for total RNA extraction for RT-PCR verification. The
results
showed that genomic amplification using primers F: gcccttcctccctttatttc and R: ctgacatcgacaccaacgat produced
two
distinct bands on a 1% agarose gel, with lengths of 711 bp and 955 bp (Fig 5-3. (c)). RT-PCR on RNA using the
same
primers yielded a distinct band of 80 bp on a 3% agarose gel (Fig 5-3. (d)), demonstrating that the T-DNA
fragment had
been inserted into the *Metarhizium* genome and was able to stably transcribe dsRNA.

![Figure 5-3. Schematic representation of the verification process](https://static.igem.wiki/teams/5588/results/5-3.webp)
![Figure 5-3. Schematic representation of the verification process](https://static.igem.wiki/teams/5588/results/5-4.webp)

Fig 5-3.
Validation results of *Metarhizium* engineered via Agrobacterium-mediated transformation for dsF3
production.
(a) Agarose gel electrophoresis results of PCR verification for *Agrobacterium* transformed with the
pBar\_PtrpC-dsF3-TtrpC plasmid. M, DNA molecular weight marker (from top to bottom: 2000 bp, 1000 bp, 750 bp,
500 bp, 300 bp, 100 bp). 1–5, PCR results of transformed *Agrobacterium* AGL1 colonies. +, PCR result
of the
positive control plasmid. −, negative control. (b) Growth of transformed *Metarhizium* after transfer
from
M-100 medium to SDAY medium containing glufosinate. Mpbar, *Metarhizium* transformed with the empty
vector.
Mpbar-dsRNA-1 and Mpbar-dsRNA-2, *Metarhizium* transformed with the pBar\_PtrpC-dsF3-TtrpC plasmid.
(c) Agarose
gel electrophoresis results of genomic PCR for transformed *Metarhizium*. M, DNA molecular weight
marker
(from top to bottom: 2000 bp, 1000 bp, 750 bp, 500 bp, 300 bp, 100 bp). 1, Genomic PCR of Mpbar-dsRNA-1. 2,
Negative control for Mpbar-dsRNA-1. 3, Genomic PCR of Mpbar-dsRNA-2. 4, Negative control for Mpbar-dsRNA-2.
5, Genomic PCR of wild-type *Metarhizium*. 6, Negative control for wild-type *Metarhizium*. +,
PCR result of the
positive control plasmid. (d) Agarose gel electrophoresis results of RT-PCR using total RNA extracted from
successfully transformed *Metarhizium*. M, DNA molecular weight marker (from top to bottom: 1000 bp,
700 bp,
500 bp, 400 bp, 300 bp, 200 bp, 100 bp). 1–5, RT-PCR results of Mpbar-dsRNA-1. −, RT-PCR result of
wild-type *Metarhizium*.

### Phenotypic Results of Infection by Engineered *Metarhizium*

To further confirm the virulence of the modified *Metarhizium* against brown citrus aphids, we scaled
up the
culture of the verified successfully transformed strains to prepare spore suspensions. The spore suspension
was
adjusted to a concentration of 1×10⁷ spores/mL. Tender citrus branches were placed in EP tubes filled with
distilled water and fixed inside 50 mL centrifuge tubes. Brown citrus aphids were immersed in the spore
suspension for 15 seconds, then transferred to the tender branches for cultivation.

It was observed that after 3 days, white mycelia grew on the aphid body surface and the aphids’ movement was
restricted.
After 6 days, the aphids died, the mycelia decreased, and green spores began to appear on their body surface.
After 9
days, the aphid cadavers were fully covered with spores (see Fig 5-4.).

![Figure 5-4 Part 2](https://static.igem.wiki/teams/5588/results/img-v3-02qs-3e268441-f240-41da-980f-6a5199d7991g.avif)

Fig 5-4.
Phenotypic results of brown citrus aphids infected by engineered *Metarhizium*. (a) Physiological
state of
brown citrus aphids without *Metarhizium* infection.(b) Physiological state of brown citrus aphids 3
days
after infection with spores of the engineered *Metarhizium*.(c) Physiological state of brown citrus
aphids 6
days after infection with spores of the engineered *Metarhizium*.(d) Physiological state of brown
citrus
aphids 9 days after infection with spores of the engineered *Metarhizium*.

![](https://static.igem.wiki/teams/5588/page/linehead2.webp)

## Discussion

There are various types of natural fungi capable of infecting brown citrus aphids; however, their infection
process takes too long, resulting in low pathogenicity against brown citrus aphids. Currently, the control
effect of fungal preparations on brown citrus aphids is mostly unsatisfactory. Therefore, **it is of
great
significance to improve the pathogenicity of fungal strains against brown citrus aphids through molecular
approaches**.

*Metarhizium anisopliae* was genetically modified via Agrobacterium-mediated
transformation to express dsRNA that silences the CHS, CYP450, and CP19 genes of brown citrus aphids during
infection, thereby enhancing the pathogenicity of the transgenic *Metarhizium* against brown citrus
aphids. In
this study, we successfully characterized the expression of this dsRNA and the phenotypic phenomena of brown
citrus aphids infected by the engineered *Metarhizium*. However, due to time constraints, we aim to
characterize
the silencing effect of the transgenic *Metarhizium* on the three key genes of brown citrus aphids and
determine
its stability and biological activity in future research.

Based on the design of a responsible project, we
expect to establish a model of the engineered *Metarhizium* biopesticide spreading in the field and
infecting
aphid populations, **which simulates the process of the fungus spreading from treated plants to adjacent
plants and causing infections**. This result will provide guidance for farmers to determine the
optimal
spraying time, spraying location, and spraying dosage, enabling efficient, economical, and environmentally
friendly aphid control. For detailed modeling, please refer to the "model" section.

Considering **the
safety issues of genetically modified microorganisms (GMOs) in application**, we designed a safety
switch system by referring to the designs of the previous NYMU\_Taipei team and Hangzhou-SDG team and combining
our own needs to ensure the safety of the engineered fungi. For detailed design, please refer to the "design"
section.

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# Overlook

This year, the SZU-igem team has verified through multi-module experiments that the Aphigo Intelligent Citrus
Aphid
Management System is practical and valuable. The detection module uses engineered *Bacillus subtilis*
to convert
aphid
honeydew signals into quantifiable MeSA gas, solving the "difficulty in detection"
problem; the RNAi
therapy module, after four generations of improvements, has increased the aphid mortality rate from 31.4% to
**60.2%**, and
the optimized system is conducive to **large-scale production**; the delivery system uses MS2
VLPs as carriers to ensure the stable and efficient delivery of RNAi molecules; the fungal
contact-killing module
enables engineered *Metarhizium* to play a dual role, providing targeted prevention and control during
the aphid
outbreak
period.

These achievements form a complete governance process of "**early warning triggering → RNAi defense
activation
→ fungal
enhanced elimination**," breaking through the limitations of traditional chemical pesticidesand
conventional biological control. This system helps citrus growers achieve precise
pest
control, reduce costs and risks, and provides an innovative model of "synthetic biology + digital agriculture"
for the
green management of agricultural pests and diseases, thereby promoting the sustainable transformation of the
citrus
industry.

In the future, we are committed to transforming this innovative achievement into broader and deeper practical
applications. With technology as the brush and green development as the ink, we will paint a new chapter of
ecological
harmony and industrial prosperity on the canvas of global agriculture, **making the Aphigo Intelligent
Management System a
solid shield for safeguarding global agricultural ecological security and food security.**

![](https://static.igem.wiki/teams/5588/page/linehead1.webp)

# References

Click to EXPAND the content

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