iGEM 2023 — Team szu-china

{% extends "layout.html" %} {% block page\_content %}

## Notebook

[ ]

# Menu

[ ]

[Overview](#anchor1)

[ ]

[Team log](#anchor2)

[Brain stroming](#anchor2.1)

[Wet lab](#anchor2.2)

[Products](#anchor2.3)

[Dry lab](#anchor2.4)

[Hardware](#anchor2.5)

[Human practice](#anchor2.6)

[Art desigining](#anchor2.7)

[Wiki Develop](#anchor2.8)

## Overview

#### 2022.12.3 Set up a team

We set up a team with 13 members.

#### 2022.12.16-2023.2.6 Experimental skills training and literature reading training

#### 2023.1.16-3.19 Brain storming

Brain storming was carried out in groups. Each group had its own
project.

#### 2023.3.20 Determined our project

The investigation in Haijixing Market triggered our interest in
*Botrytis cinerea*.

#### 2023.3.20-2023.5.12 Background research and preliminary experiments

The preliminary design of the complete project system and a large
number of background research and literature research were
completed. Some experiments were started.

#### 2023.8.14-2023.10.2 Middle period of experiments and product iteration

#### 2023.10.12 Finished all experiments and uploaded our wiki

#### 2023.11.2 iGEM jamboree

## Team log

### 2023.1.16-3.19 Brain stroming

#### 2023.1.16 Start our brain storming

1. Discuss and determine the complete process of team
brainstorming and establish the "three-step" strategy:

Step 1. Release your thoughts and record them.

Step 2. Search and organize a lot of data.

Step 3. Initially select the project with the most development
potential for further advancement, and finally select the most
suitable one as our project after reviewing each pre-project
from multiple perspectives.

2. Created a brainstorming summary sheet to record our
interesting ideas.

#### 2023.1.16-2023.3.20 Built some brain storming

#### Therapeutics

Yutong Chen——Phages control plant wilt.

Xinxu Long——Treatment of gluten sensitivity.

Tangkun Zhu——Body fluids detection for breast cancer.

#### Environment

Yutong Chen——Prevention and control of Fushou snails.

#### Manufacturing

Yutong Chen——Produce chitosan to preserve fresh fruits and
vegetables.

Zuxin Chen——A damage-free bleaching system.

Jun Huang——Flavors are produced with human metabolites.

#### Food and Nutrition

Xiaocheng Feng,Yutong Chen——Remove nitrite from leftovers.

#### Energy

Yutong Chen——Monitoring of soil moisture.

#### 2023.3.20 Change of brain storming

The investigation in Haijixing Market triggered our interest in
Botrytis cinerea.

#### 2023.4.13 Determined our project

Listened to our PI's opinions and two control measures, RNAi and
plant immunity were formed.

### 2023.4.13-2023.10 Work implementation

### Wet lab

#### RNAi

#### Period one,transformation, colony PCR, plasmid extraction, plasmid PCR, and infection experiments attempted

#### 5.22-5.23

Xinxu Long,Ruoyu Chen——Transformation of RNAi plasmids;
Inoculation of gray mold fungus onto PDA agar plates.

#### 5.24-5.27

Xinxu Long,Ruoyu Chen——Plasmid extraction; Colony PCR of
*HT115(DE3)* and subsequent gel electrophoresis analysis.

#### 5.28-5.29

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with fungal agar plugs, inoculation with
fungal spore suspension); Inoculation of gray mold fungus.

#### 5.30

Xinxu Long——Plasmid extraction; Plasmid PCR and gel
electrophoresis analysis of *HT115(DE3)*.

#### 6.1

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with circular filter paper soaked with
fungal spore suspension); Scratch inoculation of gray mold
fungus on agar plates for sporulation induction.

#### 6.5-6.7

Xinxu Long,Xiaocheng Feng——Transformation of RNAi plasmids;
Plasmid extraction; Colony PCR and gel electrophoresis analysis
of
*HT115(DE3)*; Plasmid PCR and gel electrophoresis analysis
of *HT115(DE3)*.

#### 7.5

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits and detached leaves, with four different infection
methods attempted: agar plug inoculation, glass paper with gray
mold, fungal spore suspension, circular filter paper soaked with
fungal spore suspension.

#### Period two,shRNA extraction, infection experiment attempts

#### 7.11-7.18

Ruoyu Chen,Xinxu Long,Xiaocheng Feng——Inoculation of gray mold
fungus onto PDA agar plates; Shake culture, induction of
Escherichia coli with IPTG, extraction of various shRNAs using
Trizol method and completion of nucleic acid gel
electrophoresis.

#### 7.19-7.20

Xinxu Long,Xiaocheng Feng——Inoculation of gray mold fungus onto
PDA agar plates; Shake culture, induction of Escherichia coli
with IPTG, extraction of various shRNAs using Trizol method and
completion of nucleic acid gel electrophoresis.

#### 7.21

Xinxu Long,Xiaocheng Feng——Bulk extraction of shRNA and
completion of nucleic acid gel electrophoresis; Gray mold fungus
infection on tomato fruits and detached leaves (inoculation with
agar plugs); Inoculation of gray mold fungus onto PDA agar
plates.

#### 7.22-7.24

Xinxu Long,Xiaocheng Feng——Bulk extraction of shRNA and
completion of nucleic acid gel electrophoresis.

#### 7.25

Xinxu Long,Xiaocheng Feng——After spraying with naked shRNA or
CPP-coated shRNA, tomato fruits were inoculated with gray mold
fungus using agar plugs; Transformation of GFP plasmid;
Inoculation of gray mold fungus onto PDA agar plates.

#### 7.26-7.28

Xinxu Long,Xiaocheng Feng——Bulk extraction of shRNA and
completion of nucleic acid gel electrophoresis; Scratch
inoculation of gray mold fungus on agar plates for sporulation
induction; Inoculation of gray mold fungus onto PDA agar plates.

#### 7.29

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with fungal spore suspension); Inoculation
of gray mold fungus onto PDA agar plates.

#### 7.30

Xinxu Long,Xiaocheng Feng——Bulk extraction of shRNA and
completion of nucleic acid gel electrophoresis.

#### 8.1

Xinxu Long,Xiaocheng Feng——Attempted to obtain gray mold spores
and prepare spore suspension; Observed spore morphology under a
microscope; Tried rapid sporulation method; Simulated natural
gray mold infection on tomato fruits using a 5x4 egg carton.

#### 8.2

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with fungal spore suspension and mycelial
suspension); Bulk extraction of shRNA and completion of nucleic
acid gel electrophoresis.

#### 8.3

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with fungal spore suspension); Bulk
extraction of shRNA and completion of nucleic acid gel
electrophoresis.

#### 8.4

Xinxu Long,Xiaocheng Feng——Inoculation of gray mold fungus onto
PDA agar plates; Bulk extraction of shRNA and completion of
nucleic acid gel electrophoresis.

#### 8.5

Xinxu Long,Xiaocheng Feng——Gray mold fungus infection on tomato
fruits (inoculation with fungal spore suspension); Bulk
extraction of shRNA and completion of nucleic acid gel
electrophoresis.

#### 8.8

Xiaocheng Feng——Inoculation of gray mold fungus onto PDA agar
plates; Bulk extraction of shRNA and completion of nucleic acid
gel electrophoresis.

#### Period three,application of naked shRNA

#### 8.10-8.11

Xiaocheng Feng,Jun Huang,Yutong Chen——After spraying with naked
shRNA, tomato fruits were inoculated with small agar plugs of
gray mold fungus.

#### 8.12-8.14

Xiaocheng Feng,Jun Huang——Processing of lesion area data;
Inoculation of gray mold fungus onto PDA agar plates.

#### 8.15-8.16

Xiaocheng Feng,Jun Huang——After spraying naked shRNA, tomato
fruits were inoculated with small agar plugs of gray mold
fungus.

Xiaocheng Feng——Bulk extraction of shRNA and completion of
nucleic acid gel electrophoresis.

#### 8.17

Xiaocheng Feng——Bulk extraction of shRNA and completion of
nucleic acid gel electrophoresis.

Yutong Chen——Inoculation of gray mold fungus onto PDA agar
plates.

#### 8.18

Xiaocheng Feng,Jun Huang——qPCR tomato sample collection.

#### 8.19

Jun Huang——Inoculation of gray mold fungus onto PDA agar plates.

Xiaocheng Feng,Jun Huang,Yutong Chen——qPCR tomato sample
collection; Bulk extraction of shRNA and completion of nucleic
acid gel electrophoresis.

Xiaocheng Feng——Scratch inoculation of gray mold fungus on agar
plates for sporulation induction; Microscopic observation of
spore morphology.

#### Period four,application of shRNA coated with CPP (Cell Penetrating Peptide)

#### 8.23

Xiaocheng Feng,Jun Huang——After spraying with shRNA coated with
CPP (Cell Penetrating Peptide), tomato fruits were inoculated
with small agar plugs of gray mold fungus.

Ruoyu Chen——Bulk extraction of shRNA.

#### 8.24

Xiaocheng Feng,Jun Huang——After spraying with shRNA coated with
CPP (Cell Penetrating Peptide), tomato fruits were inoculated
with small agar plugs of gray mold fungus.

Yutong Chen——Inoculation of gray mold fungus onto PDA agar
plates; Bulk extraction of shRNA.

#### 8.26-8.27

Jiayi Huang——qPCR tomato sample collection.

Jun Huang——Inoculation of gray mold fungus onto PDA agar plates.

#### 8.28

Xiaocheng Feng,Jun Huang,Xinxu Long——After spraying with shRNA
coated with Cell Penetrating Peptide (CPP), tomato fruits were
inoculated with small agar plugs of gray mold fungus.

Yutong Chen——Inoculation of gray mold fungus onto PDA agar
plates.

#### 8.29-9.2

Xiaocheng Feng,Xinxu Long——Sample collection of tomato samples
treated with naked RNA; RNA extraction from tomato samples;
Reverse transcription of RNA into cDNA; Primer selection;
qRT-PCR (quantitative reverse transcription PCR).

#### 9.4

Xiaocheng Feng,Xinxu Long——qRT-PCR (naked RNA treatment).

#### 9.5

Xiaocheng Feng,Xinxu Long——Bulk extraction of shRNA.

#### 9.6-9.14

Xiaocheng Feng,Xinxu Long——RNA extraction from tomato samples
(RNA treatment with CPP coating); Reverse transcription of RNA
into cDNA; qRT-PCR.

#### 9.15-9.16

Xiaocheng Feng,Xinxu Long——After spraying with shRNA coated with
CPP (Cell Penetrating Peptide), tomato fruits were inoculated
with small agar plugs of gray mold fungus.

#### Period five,Validation of concatenated shRNA.

#### 9.17

Xiaocheng Feng,Xinxu Long——RNA extraction from tomato samples
(treated with concatenated RNA coated with CPP); Reverse
transcription of RNA into cDNA; qRT-PCR; Repeat experiment -
Spraying with naked shRNA followed by inoculation of tomato
fruits with small agar plugs of gray mold fungus.

#### 9.18-9.19

Xiaocheng Feng,Xinxu Long——qPCR of concatenated RNA.

#### 9.20-9.28

Xiaocheng Feng,Xinxu Long——For continuous qPCR (shRNA stability
test) infection experiments; Sample RNA, cDNA reverse
transcription and qPT-PCR for continuous qPCR and bare shRNA
were extracted.

#### Plant Immunity

#### 5.18-5.27

Yutong Chen——Construction of PehA Plasmid

#### 5.31-6.4

Ruoyu Chen,Tangkun Zhu——Transformation of PehA plasmid and
inoculation of transformed *BL21 (DE3)* onto agar plates.

#### 6.5-7.15

Yutong Chen,Tangkun Zhu——Plasmid extraction for PCR and enzyme
digestion, followed by gel electrophoresis verification.

#### 7.16-7.30

Yutong Chen,Tangkun Zhu——IPTG induction, extraction of PehA
protein, protein concentration determination using the BCA
method, protein electrophoresis using SDS-PAGE, and Western blot
verification of PehA expression.

#### 7.24-7.31

Yutong Chen,Tangkun Zhu,Yang Yang——Two methods, protoplast
accumulation and extracellular alkalinization, were used to
identify the immune response induced in leaves after flg22
application.

Ruoyu Chen——Construction of BvEP plasmid.

#### 7.31-8.1

Ruoyu Chen,Tangkun Zhu——Activation of
*BL21 (DE3)* containing the flg22 plasmid and inoculation
onto agar plates.

#### 8.2

Yutong Chen——Extraction of flg22 plasmid for PCR and enzyme
digestion, followed by gel electrophoresis verification.

#### 8.3-8.20

Yutong Chen,Tangkun Zhu——IPTG induction, extraction of flg22
protein, protein concentration determination using the BCA
method, protein electrophoresis using SDS-PAGE, and Western blot
validation of flg22 expression.

#### 8.4-9.24

Yutong Chen,Tangkun Zhu,Yang Yang——DAB staining method for
identifying the ROS burst in leaves induced by flg22
application.

#### 8.9

Ruoyu Chen,Tangkun Zhu——Transformation of BvEP plasmid and
inoculation of the transformed *BL21 (DE3)* onto agar
plates.

#### 8.10-8.20

Yutong Chen,Tangkun Zhu——IPTG induction, extraction of BvEP
protein, protein concentration determination using the BCA
method, protein electrophoresis using SDS-PAGE, and Western blot
validation of BvEP expression.

#### 8.21

Yutong Chen——Optimization of IPTG induction conditions for the
expression of flg22 and BvEP.

#### 8.22-9.24

Yutong Chen,Yang Yang——New IPTG induction, extraction of flg22
and BvEP proteins, protein concentration determination using the
BCA method, protein electrophoresis using SDS-PAGE, and Western
blot validation of flg22 and BVEP expression.

#### 9.21

Yutong Chen,Tangkun Zhu,Yang Yang——Identification of ROS burst
in leaves after application of PehA, flg22, and BvEP using
luminescent staining method.

#### 10.2

All experiments have concluded.

### Products

#### 8.15

Tangkun Zhu, Ruoyu Chen——Summarize the current situation of
pesticide use, including pesticide abuse and the use of RNAi
biopesticides. Gets the inspiration of making the product into a
laundry detergent beads from our PI. We our initial idea is to
make our product into a bead.

#### 8.25

Tangkun Zhu——Learns how the laundry detergent beads produce,
finding the raw material for bead production.

#### 9.10

Xinxu Long, Tangkun Zhu, Ruoyu Chen——We interview Mr. Xiang and
Mr. Zhang from the Testing Center of Shenzhen University to
discuss what kind of electron microscopy can be used to observe
our biological samples.

#### 9.13

Tangkun Zhu——Designs our product shapes, content and
accessories. We decided to use PVA (polyvinyl alcohol) film as
the material for our packaging.

#### 9.14

Xinxu Long, Tangkun Zhu, Ruoyu Chen——After learning and
comparing the use rules and observation objects of different
electron microscopy, we decide to use scanning electron
microscopy (SEM) to observe our RNA-CPP complex at the
microscopic scale. We designed CPP, RNA and RNA-CPP complexes
with different concentrations and observed them under SEM.

#### 9.21

Xinxu Long, Tangkun Zhu, Ruoyu Chen——Use scanning electron
microscopy (SEM) to observe our RNA-CPP complex at the
microscopic scale. We use SEM for further observation and
determine the structure of our sample under SEM.

#### 9.24

Tangkun Zhu——Finds the most suitable solvent contained in our
complex biopesticide products.

#### 9.26-10.5

Tangkun Zhu——Designs the experiment of product verification and
carry out the related test, and carry out the product
production.

### Dry lab

#### 7.11-8.5

Jun Huang——Building an RNA interference model.

Jun Huang——Building an epidemiological model. The model guides
how the product is applied.

#### 8.5-9.3

Jun Huang——Helped with tomato infestation tests in the lab.

Jun Huang——Analyzing the data from the experiment.Data were
analyzed to advance the experiment.

#### 9.10-9.20

Jun Huang——validating the model against the data produced by the
experiment.

### Hardware

#### 5.1-6.31

Zhonghao Sun——Conception of shRNA production by in vitro
transcription.

Zhonghao Sun—— Preliminary design of hardware functions and
structure.

#### 7.1-7.20

Zhonghao Sun——Model selection of hardware components and
microcontrollers.

Zhonghao Sun——3D Modeling of the mini reaction container with
3ds MAX.

#### 7.21-8.31

Zhonghao Sun ——3D Printing of the mini reaction container.

Zhonghao Sun ——Assembly of various components.

Zhonghao Sun ——Writing and debugging of microcontroller
programs.

#### 9.1-10.2

Zhonghao Sun ——Attempt of producing shRNA using the built
hardware.
Zhonghao Sun ——Verify the shRNA produced by the hardware.

### Human practice

#### March

#### 3.11

All members——Shenzhen University Affiliated Experimental High
School visited the iGEM lab for learning and exploration,
where we introduced synthetic biology to them.

#### 3.20

Zuxin Chen,Xinyi Hao——Haijixing Agricultural Market Research:
After learning about the threat of gray mold disease to
tomatoes, we initiated a brainstorming session to develop a
preliminary plan.

#### April

#### 4.2

All members——Online exchange meeting with China Agricultural
University to share our respective project brainstorms and
provide suggestions to each other.

#### 4.22

All members——Participated in the Deep Blue Charity Sale, where
iGEM teams not only raised funds for public welfare projects
but also contributed to the dissemination and promotion of
synthetic biology knowledge. This helped promote innovation,
education, and the spirit of public service.

#### 4.23

All members——Innovation and Entrepreneurship Carnival, where
entrepreneurs shared their startup stories and experiences,
providing valuable advice for our future entrepreneurial
plans.

#### May

#### 5.13

Zuxin Chen,Xinyi Hao——Interview with Mr. Li Xiaojie, Deputy
Director of the Technology Department at the Genomics
Institute. He emphasized the importance of adopting a
"prevention is better than cure" plant protection philosophy
and highlighted the significance of focusing on product
stability, delivery efficiency, and effectiveness.

#### 5.21

All members——South China Exchange Conference, which brought
together teams from the South China region to build a bridge
for interactive displays, collaborative exchanges, and mutual
learning, creating a conducive atmosphere for exploring
synthetic biology together.

#### 5.31

Zuxin Chen,Xinyi Hao——Interview with Professor Zhang Yongmin
from Shenzhen University, who believes that plant vaccines
play a crucial role in sustainable agriculture by effectively
reducing the use of pesticides on plants.

#### July

#### 7.11

All members——CCIC Conference, which brought together teams
from different regions to facilitate interactive displays,
collaborative exchanges, and mutual learning.

#### 7.11

All members——We had a discussion with the team from Zhejiang
University, exchanging ideas and discussing the project, while
providing each other with suggestions.

#### 7.16

All members——High school teams visited our lab for tours and
exchanges, followed by project presentations and discussions.

#### 7.16

All members——iGEM team from Macau University exchanged ideas,
discussing projects and planning the first iGEM Greater Bay
Area Synthetic Biology Industry-Academia-Research Forum.

#### 7.20

Zuxin Chen,Xinyi Hao——The Shenzhen Agricultural Technology
Promotion Center conducted an interview and exchange with Dr.
Li Yonghong, Chief Researcher at the Shenzhen Agricultural
Promotion Center, and Dr. Jin Man from the Chinese Academy of
Sciences. They provided us with a broader perspective and
targeted solutions.

#### 7.22

Zuxin Chen,Xinyi Hao——We conducted interviews with
agricultural markets and supermarkets, which will help us
better meet market demands and consumer expectations with our
products.

#### August

#### 8.10

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Tangkun Zhu,Jiayi Huang——We
visited the Huizhou Boluo County Agricultural Technology
Demonstration Farm to understand the usage of pesticides, the
promotion methods for new products, and the promotional
responsibilities undertaken by the department.

#### 8.11

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Tangkun Zhu,Jiayi
Huang——Director Yin from the Pesticide Residue Testing Center
of Boluo County Agricultural Bureau highly praised our
biopesticides, emphasizing the importance of gaining
recognition from farmers and ensuring effective pesticide
performance.

#### 8.11

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Tangkun Zhu,Jiayi
Huang——Interviewed Senior Engineer Wang Rifang and Teacher Li Li
from the Agricultural Comprehensive Service Center of Boluo
County Agricultural and Rural Service Center, discussing major
tomato diseases and related issues.

#### 8.12

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Tangkun Zhu,Jiayi Huang——Visited
Guangdong Hongke Testing Technology Co., Ltd., consulting them
on specific pesticide residue testing procedures and touring
their laboratory.

#### 8.15

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Tangkun Zhu,Jiayi Huang——We
conducted an interview with Technician Hong at Huizhou
BoshangJiaonong Ecological Agriculture Development Co., Ltd.
Here, we gained insights into tomato cultivation and his unique
approach to farming.

#### 8.19

Zuxin Chen,Xinyi Hao,Honglin Wei,Yang Yang,Jiayi Huang——First
iGEM Greater Bay Area Synthetic Biology
Industry-Academia-Research Forum, exploring new ways of
collaboration among iGEM teams and emphasizing the collaboration
between academia and industry.

#### 8.20

Zuxin Chen,Xinyi Hao,Ruoyu Chen,Erhuan Guan——University City
Parent-Child Summer Camp, organizing a series of carefully
planned activities to ensure meaningful experiences for the
participants.

#### September

#### 9.3

All members——2023 iGEM Intercollegiate Synthetic Biology Science
Popularization Conference, we provided detailed explanations of
our project to participating students and conducted science
popularization on the safety of biopesticides.

#### 9.10

All members——A series of Shenzhen University lectures aimed at
gradually guiding college students to deepen their understanding
of this field.

#### 9.23

Zuxin Chen,Xinyi Hao,Yang Yang,Jiayi Huang——We visited the
Dacheng Xin Qianxi Tomato Industry Base for an interview and to
conduct trials using our products.

### Art desigining

#### 3.4-3.9

Jiayi Huang,Erhuan Guan——Making original gifts and designing
game cards related to synthetic biology for students from
Affiliated High School of Shenzhen University.

#### 4.8-4.22

Jiayi Huang,Erhuan Guan——Designing materials for Shenlan
Campus Sales and annual exposition for
students'entrepreneuring and businessing.

#### 4.23-5.1

Erhuan Guan——Designing team uniforms.

#### 5.3-5.21

Jiayi Huang,Erhuan Guan——Designing the 7th Southern China
Regional Meeting related materials.

#### 7.5-7.7

Jiayi Huang,Erhuan Guan——Designing poster of our project for
CCiC(Conference of China iGEMer Community).

#### 7.10-7.20

Jiayi Huang—— Drawing emoji pics.

#### 8.1-8.9

Jiayi Huang,Erhuan Guan——designing painting albums for
education.

#### 8.10-8.16

Jiayi Huang——Recording the human practice in huizhou .

#### 8.28-9.14

Jiayi Huang——Completing promotion video.

#### 9.15-10.12

Jiayi Huang,Erhuan Guan——Shooting the presentation video and
designing the wiki.

### Wiki Develop

#### 6.20-7.15

Honglin Wei——Familiarize with the official iGEM wiki editing
operations.

#### 7.15-8.20

Honglin Wei—— learning HTML, CSS, and JavaScript.

#### 8.20-9.15

Honglin Wei——Build the wiki framework for our SZU iGEM 2023.

#### 9.15-10.12

Honglin Wei——Complete the home page and gradually complete other
necessary pages.

Thanks

A big thank you to Ling Rongsong, Wu Yingfeng, Shen Yunfeng from
Zhejiang University, and others for their help. I am extremely
grateful to the other members of SZU 2023 iGEM team and our
close collaboration, which allowed us to successfully complete
the creation of this wiki!

{% endblock %}

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

## Engineering

[ ]

# Menu

[ ]

[Overview](#anchor1)

[ ]

[RNAi Silence System](#anchor2)

[RNA V1: shRNA](#anchor2.1)

[RNA V2: BP100-(KH)9 + shRNA](#anchor2.2)

[RNA V3: BP100-(KH)9 + bi-shRNA](#anchor2.3)

[ ]

[PolycoBead](#anchor3)

[PolycoBead 1.0](#anchor3.1)

[PolycoBead 2.0](#anchor3.2)

[References](#refer)

## Overview

In our project, we have developed two systems to comprehensively
control tomato gray mold disease caused by
*Botrytis cinerea*. Our plant immune system is used to
activate the broad-spectrum immunity of tomatoes to prevent tomato
gray mold disease, while our RNAi system is used to kill the
*B. cinerea* or reduce its infectivity. To combine the two
systems, we use the polycobead system to encapsulate the plant
immune system and the RNAi system, making the biopesticide more
storage-friendly and convenient to use.

In our Project, we followed the DBTL principle, tested the
functions of RNAi and PolycoBead during the project cycle, and
improved and iterated according to the test results, so that they
finally achieved our expected results.

![](https://static.igem.wiki/teams/4653/wiki/engineering/cycle.jpg)

## RNAi Silence System

### RNA V1: shRNA

#### Design

Due to its high specificity, safety, and sustainability, we
have chosen RNAi technology for the treatment of tomato gray
mold disease. After screening, we selected *BcchsIIIa*,
*Bccyp51*, *BcOAH1*, and *Bcpme1* as our
targets for RNAi. Since shRNA is known to have higher
stability in natural environments and can be produced on a
large scale at low cost using fermentation engineering in the
laboratory, we have selected the most common strain,
*E. coli*, for fermentation production. We used
*E. coli*
HT115(DE3), which lacks nucleases. The designed target
sequences are constructed through the order of sense fragment
loop-antisense fragment, and connected to the plasmid
pET-28a(+) to obtain our shRNA.

#### Bulid

To verify our successful transformation, we used specific
primers to amplify the plasmids extracted from
*E. coli* by PCR, and demonstrated that our shRNA
expression vector successfully entered
*E. coli* HT115(DE3) by agarose gel electrophoresis
(Figure1). Subsequently, RNA was extracted from
*E. coli* after IPTG induction, and the successful
production of shRNA in *E. coli* was confirmed by the
size of the bands.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-engineering-plasmid-2.png)
**Figure 1. Agarose gel electrophoresis of plasmids after
plasmid PCR 1-5: Plasmids control; 5-10: Plasmids
extracted from *E.coli***

#### Test

In our experiments, we used shRNA(gfp) as the control
group.The produced shRNA was sprayed on the tomato fruits
infected with *B. cinerea*, and we explored the
inhibitory effect of shRNA on gray mold infection in tomatoes
by measuring the distribution of disease spots (Figure 2a). We
also detected the inhibitory effect of shRNA on target genes
using qRT-PCR (Figure 2b). The results indicate that on the
third day, the highest silencing rate of the target gene
achieved by spraying shRNA(Pme1)-2 was 59%.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-outlook1.png)
**Figure 2. (a)Distribution of disease spots of tomato
fruits after naked shRNA application.**

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-qpcr1.png)
**Figure 2. (b)Silencing rate of target gene after naked
shRNA application.**

#### Learn

We found that the inhibitory effect of shRNA on gray mold
disease was not as good as we had expected in actual use,
which may be because shRNA is still not very stable in natural
environments and the probability of cell uptake and processing
of shRNA is not high. Faced with this problem, we must find a
more effective system to deliver our shRNA.

### RNA V2: BP100-(KH)9 + shRNA

#### Design

Cell-penetrating peptides (CPPs) have been shown to penetrate
the plasma membrane of various plant species and tissues.
Multiple studies have demonstrated that CPPs can enhance the
stability of shRNA and significantly improve the efficiency of
silencing through CPP-mediated sRNA delivery systems.
Ultimately, we have chosen BP100-(KH)9 as our CPP
and allowed it to bind to shRNA, hoping that it will confer
greater stability to our shRNA under natural conditions and
enhance the efficiency of gene silencing on the target gene.

#### Bulid

To obtain the BP100-(KH)9+shRNA complex, we
incubated CPP and shRNA in a fixed ratio at room temperature.
For the binding of BP100-(KH)9 with shRNA, we
observed naked shRNA (Figure 3a), naked BP100-(KH)9
(Figure 3b), and BP100-(KH)9+shRNA complex (Figure
3c)under scanning electron microscopy (SEM), and significant
morphological differences were observed among the three. The
presence of spherical aggregates in Figure 3c suggests the
successful binding of BP100-(KH)9 with shRNA.

To learn more detailed information about shRNA,
BP100-(KH)9, and shRNA+CPP under SEM, please click
on
[Proof of Concept](https://2023.igem.wiki/szu-china/proof-of-concept)

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-wng-semcpp.png)
**Figure 3. (a)Morphological structure of naked shRNA
under scanning electron microscope at 20,000x
magnification (the arrow points to shRNA
aggregates).**

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-semrna-2.png)
**Figure 3. (b)Morphological structure of Naked CPP
under scanning electron microscope at 50,000x
magnification (the arrow points to CPP aggregates).**

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-semrna-cpp.png)
**Figure 3. (c)Morphology of shRNA+CPP under scanning
electron microscope at 40,000x magnification.**

#### Test

We sprayed shRNA complexed with BP100-(KH)9 onto
tomato fruits infected with *B. cinerea* and explored the
inhibitory effect of BP100-(KH)9+shRNA on gray mold
disease by measuring the distribution of disease spots (Figure
4a). We also detected the inhibitory effect of
BP100-(KH)9+shRNA on the target gene using qRT-PCR
(Figure 4b). From the results, it can be observed that
BP100-(KH)9+shRNA significantly enhanced the
efficiency of shRNA-mediated gene silencing on the target
gene. Among them, BP100-(KH)9 showed the highest
increase in silencing rate against *Bccyp51*. The
silencing rate of
*Bccyp51*
increased from 51% to 66%.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-outlook2.png)
**Figure 4. (a)Distribution of disease spots of tomato
fruits after BP100-(KH)9 + shRNA
application.**

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-qpcr2.png)
**Figure 4. (b)Silencing rate of target gene after
BP100-(KH)9 + shRNA application.**

Furthermore, through continuous qRT-PCR analysis, we found
that the highest silencing rate of the target gene by
BP100-(KH)9+shRNA was achieved at the 60th hour
(Figure 5).

For more information on continuous qRT-PCR, please refer to
[Proof of Concept](https://2023.igem.wiki/szu-china/proof-of-concept).

Additionally, the time-dependent efficiency of
BP100-(KH)9+shRNA-mediated gene silencing observed
in the experiment is consistent with our RNAi model.

For more information on the model, please see the
[Model](https://2023.igem.wiki/szu-china/model)

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-q.png)
**Figure 5. Variation in silencing efficiency of target
gene by BP100-(KH)9+shRNA over 12-96 hours.**

#### Learn

Although it has been demonstrated that
BP100-(KH)9+shRNA-mediated gene silencing of
individual target genes has shown promising efficacy in the
treatment of tomato gray mold disease, we are still not
entirely satisfied with the inhibitory effect of a single
shRNA on the infection of
*B. cinerea*, especially the unsatisfactory silencing
effect on the *BcchsIIIa* gene. We believe that this
situation may be attributed to instability of a single shRNA,
among other factors, ultimately limiting the optimal
effectiveness of single-target gene silencing by shRNA.

Moreover, in order to achieve better suppression of gray mold,
a large amount of single shRNA is required, which also reminds
us to always consider the cost issue of shRNA.

### RNA V3: BP100-(KH)9 + bi-shRNA

#### Design

We have learned that the bi-shRNA design leads to more rapid
onset of gene silencing, higher efficacy, and greater
durability when compared with either siRNA or conventional
shRNA. By concatenating shRNA molecules targeting different
mRNA sequences, the resulting shRNAs can be processed into
different siRNAs that act simultaneously. Moreover, compared
to mixing and spraying different shRNAs, by concatenating
different shRNAs to obtain bi-shRNA, we can save more on the
usage of shRNAs while achieving better silencing effects. As
shown in the Figure 6, we used website prediction again to
optimize shRNAs targeting *BcchsIIIa* and *Bccyp51*,
resulting in shRNA(CHSIIIa)\* and shRNA(cyp51)\*, respectively.
We then concatenated them to construct bi-shRNA named
Box-survival, which collectively silences two important genes
essential for the survival of *B. cinerea*. During
application, we will also combine it with cell-penetrating
peptide BP100-(KH)9 to construct
BP100-(KH)9+bi-shRNA for optimal inhibitory
effects.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-bs.png)
**Figure 6. The design of our Box-survival(bi-shRNA).**

#### Bulid

To validate the successful production of Box-survival in
*E. coli*, we constructed a plasmid and transformed it
into *E. coli* for large-scale cultivation. After
induction with IPTG, we extracted RNA from *E. coli* and
confirmed the successful generation of shRNA based on the band
size observed on the gel. In the gel image, there is a bright
band between lanes 100 and 150, which corresponds to the size
of 124bp. This is because Box-survival is a concatenation of
two shRNAs, resulting in a larger size compared to the
designed single-target shRNA (69bp), showing a significant
molecular weight difference.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-iptg.png)
**Figure 7. Gel electrophoresis of RNA extracted from
*E. coli*HT115(DE3).
shRNA(Box-survival) exhibits a significantly larger
molecular weight.**

#### Test

Similarly, we sprayed BP100-(KH)9 + bi-shRNA on
tomato fruits infected with *B. cinerea* and compared the
inhibitory effects of BP100-(KH)9 + bi-shRNA with
our selected four single-target BP100-(KH)9 +
bi-shRNA by measuring the distribution of disease spots
(Figure 8a). We also examined the inhibitory effect of
BP100-(KH)9 + bi-shRNA on the target genes of
*B. cinerea* using qRT-PCR, comparing it with
BP100-(KH)9 + shRNA (Figure 8b). From the results,
it can be observed that the optimized sequences of
shRNA(CHSIIIa)\* and shRNA(cyp51)\* exhibit lower off-target
probabilities compared to the original sequences. Furthermore,
compared to BP100-(KH)9 + shRNA, BP100-(KH)9
+ bi-shRNA demonstrate a stronger inhibitory effect on gray
mold disease. From the Figure 8b, we can see that among all
shRNAs, bi-shRNA(Box-survival) showed the highest silencing
efficiency against the target gene, reaching 70%.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-outlook3.png)
**Figure 8. (a)Distribution of disease spots of tomato
fruits after BP100-(KH)9 + bi-shRNA and
BP100-(KH)9 + shRNA application.**

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-eng-qpcr3.png)
**Figure 8. (b)Silencing rate of target gene after
BP100-(KH)9 + bi-shRNA and BP100-(KH)9
+ shRNA application.**

#### Learn

According to the results mentioned above, our BP100-(KH)9
+ bi-shRNA has shown excellent inhibitory effects on the
target gene with lower off-target effects. Therefore, we can
ensure that BP100-(KH)9 + bi-shRNA can be used as
our RNAi product for field spraying, achieving precise,
efficient, and powerful attack on pathogenic fungi. The
engineering cycle of our RNAi silencing system also concludes
after this validation step.

## PolycoBead

### PolycoBead 1.0

#### Design

In order to address the instability of RNAi biopesticides and
regulate the amount of pesticide used, we hope to package our
product in a specific form to avoid misuse. We drew
inspiration from the design of laundry pods and learned that
they are made up of individual beads wrapped in PVA film.
Through investigation, we found that PVA film has good water
solubility, biocompatibility, degradability, high mechanical
strength, ease of processing, low production cost, and easy
availability. It also has barrier and anti-static properties,
which can effectively prevent RNA from coming into contact
with nucleases in the environment and being degraded.
Therefore, we decided to use PVA film to wrap our CPP-shRNA
formulations and the engineered bacterial host
*B. subtilis*.

As the produced CPP-shRNA complexes exist in the form of an
aqueous solution, we need to overcome the water solubility of
PVA. To achieve this, we plan to use a powder formulation with
strong water-absorbing capability, which does not affect the
stability of the CPP-RNA complex, to encapsulate CPP-shRNAs
within the beads. Our initial consideration is agarose, a
commonly used inert material for nucleic acid electrophoresis
in laboratories. Agarose is able to absorb water without
introducing large charged groups, thus maintaining the
stability of nucleic acids and meeting our basic requirements.
Our engineered bacteria are in the form of bacterial powder,
and users are required to add sucrose for reconstitution.

In our design of Polycobead 1.0, we mix the CPP-shRNA aqueous
solution with agarose commonly used in laboratories, and then
combine it with the engineered bacterial powder before
packaging them in water-soluble PVA film. The resulting pods
enhance the stability of the CPP-shRNA complexes and can
dissolve rapidly in water for direct application.

#### Bulid

We drop a certain concentration of CPP-shRNA aqueous solution
onto agarose powder, and then mix the engineered bacterial
powder of *B. subtilius* with the agarose powder.
Finally, we package these mixed powders into a certain size of
PVA film, and seal them using a heat sealing machine,
resulting in our Polycobead 1.0 (Figure 9).

![](https://static.igem.wiki/teams/4653/wiki/polycobead/bead.png)
**Figure 9. PolycoBead 1.0**

#### Test

To understand the structure of our CPP-shRNAs in aqueous
solution, we observed the bare shRNAs, bare CPPs and
CPP-shRNAs by scanning electron microscopy (SEM). From the
scanning electron microscope (SEM) results, it can be seen
that the naked shRNAs samples showed white spots of different
sizes under the scanning electron microscope, which may be
caused by the aggregation of shRNAs and the concentration of
aggregation is different. And at higher magnification, the
white spots will disappear more as the magnification
increases, probably because the electron energy of the
electron beam is too strong.

![](https://static.igem.wiki/teams/4653/wiki/polycobead/szu-eng-semshrna.png)
**Figure 10. naked shRNAs under SEM**

Samples of bare CPPs were examined under a scanning electron
microscope and appeared as dotted white spots in the field of
view. We speculate that these white spots are aggregates of
our cell-penetrating peptides.

![](https://static.igem.wiki/teams/4653/wiki/polycobead/szu-eng-semcpp-2.png)
**Figure 11. CPP under SEM**

Finally, our CPP-shRNAs samples were observed by scanning
electron microscopy (SEM). Many spherical polymers of
different sizes were observed in the experiment, and smaller
clumps of spherical polymers were observed after magnifying
individual polymers.

![](https://static.igem.wiki/teams/4653/wiki/polycobead/sem-cpp-rna2.png)
**Figure 12. CPP-shRNA under SEM**

At the same time, in order to test that our PVA film can
dissolve smoothly in water and release our RNAi pesticide, we
put our prepared coagulation beads into water, and the results
proved that the beads dissolved in water after about 1min. The
solution in the beads is released.

![](https://static.igem.wiki/teams/4653/wiki/polycobead/bead-in-water.png)
**Figure 13. (a)The PolycoBead 1.0 were put into the
water.**

![](https://static.igem.wiki/teams/4653/wiki/polycobead/bead-in-water2.png)
**Figure 13. (b)The PolycoBead 1.0 dissolved in water and
released contents.**

#### Learn

It is necessary to improve the release efficiency of
bio-pesticides and simplify the use of pesticides. From the
design and testing results of our PolycoBead1.0, we identified
the following limitations:

* 1. As a water-absorbing material, agarose cannot be
  completely dissolved in water at room temperature. The water
  insolubility of agarose leads to the formation of colloid
  that can cross-link CPP-shRNAs, resulting in the deposition
  of agarose at the bottom or the formation of insoluble
  precipitates after spraying on farmland, resulting in the
  inability to completely release the active ingredients of
  pesticides.

* 2. Because we added a suicide switch in the engineered
  bacteria and the strain needed to be resuscitating, the
  farmers needed to add additional sucrose to survive and
  function, which caused a certain amount of inconvenience.

* 3. The price of agarose is expensive. If it is used as an
  auxiliary material in the coagulation beads, the cost of a
  single bead will be increased, which makes it difficult to
  promote in the market.

In order to solve the above problems, we decided to iterate on
the auxiliary components of coagulation beads to give users a
more effective, convenient and affordable experience.

### PolycoBead 2.0

#### Design

Due to the disadvantages of agarose in PolycoBead1.0, we wanted
to find a liquid material that could maintain the stability of
CPP-shRNA, at the same time be soluble in water, and also would
not dissolve the PVA film. Glycerol is a polar solvent commonly
used to preserve biological samples, which can reduce the
activity of water molecules and protect biomolecules from
degradation and deformation. Glycerol can be dissolved in any
proportion with water, which ensures that the beads can release
the CPP-shRNAs complex at the fastest rate after entering the
water. In addition, although a small amount of the
glycerol-water mixture is introduced into the soil, it can also
be degraded by the microbial community. All of the above
advantages of glycerol make it the preferred choice for
providing a liquid environment in coagulation beads.

In addition, calcium alginate embedding was used to guarantee
the biological activity of *Bacillus subtiliis*
for improving the resuscitation of engineering bacteria in
PolycoBead1.0. This is one of the most widely used cell fixation
methods, which has the advantages of high density of fixed
cells, low toxicity to microorganisms, simple fabrication
process and high product release rate. By using calcium alginate
embedding method, engineered bacterial solution and sucrose can
be encapsulated in a bead at the same time. In the presence of
sucrose, the toxin-antitoxin mechanism in the engineered
bacteria is balanced to survive.

Combining the above two points, we packaged the CPP-shRNA
dispersed in glycerol system and the engineered
*B. subtilis* embedded in calcium alginate with PVA film to
form our product PolycoBead 2.0.

#### Bulid

The engineering bacteria, sucrose and 1% sodium alginate
solution were mixed and dropped into Ca2+
solution. The sodium alginate reacted with calcium ions to
form a water-insoluble calcium alginate shell in the outer
layer, while the engineering bacteria and sucrose were wrapped
in calcium alginate to form smaller beads. These beads were
added to glycerol containing CPP-shRNAs, encapsulated in PVA
films of certain size, and finally sealed using a heat sealer
to obtain our Polycobead 2.0.

**The production process of PolycoBead 2.0.**

#### Test

To ensure that the activity of CPP-shRNA could be maintained
in the glycerol system, we used the glycerol system containing
CPP-shRNA to verify by agarose gel electrophoresis. As shown
in the figure, the lanes from left to right are Marker, naked
shRNA aqueous solution, naked shRNA glycerol-aqueous solution
(glycerol: water = 9:1), CPP-shRNA complex aqueous solution,
CPP-shRNA complex glycerol-aqueous solution (glycerol: water =
9:1), and Marker repectively. It can be seen from the second
and third lanes that glycerol has a certain blocking effect on
naked shRNA, but it does not completely affect its movement in
electrophoresis. Showing in the fourth and fifth lanes, after
combining with CPP, shRNA would form a certain complex
structure with CPP and could not move in electrophoresis,
while mixing with glycerol did not affect the binding between
the two. Meanwhile, we also verified by SEM that the structure
of CPP-shRNA in glycerol system does not change.

![](https://static.igem.wiki/teams/4653/wiki/engineering/szu-engineering-polycobead-1.jpg)
**Figure 15. Agarose gel electrophoresis and SEM verified
that glycerol had no effect on CPP-shRNA.**

To demonstrate that when the calcium alginate-engineered
bacteria complex is released into the environment, the
engineered bacteria in the complex will release into the soil
at a slow rate along with the loss of Ca2+, and we
have verified that the calcium alginate-engineered bacteria
could release the liquid in the soil within 40h. We hope that
PolycoBead2.0 can be applied by simply stirring it in water,
and the effective constituent will be released to work. The
release time of PolycoBead 2.0 was tested and it could be
released in 5L water within 2min.

Click
[Proof of concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D)
for more details.

Finally, we also tested that the effective constituent in
PolycoBead 2.0 could play a good role, so we sprayed the
solution with PolycoBead 2.0 on tomatoes infected with
*B. cinerea*, and it could be obviously observed that the
solution could play a good inhibitory effect on
*B. cinerea*.

![](https://static.igem.wiki/teams/4653/wiki/poc/tomato-fruit-d1-d8.jpg)
**Figure 16. The effect of PolycoBead 2.0 was tested on
infected tomatoes.**

#### Learn

It can be concluded from the above results that our PolycoBead
can stably encase CPP-shRNA and engineered *B. subtilis*,
then release it stably after dissolving in water, and play a
good role in tomato infected with *B. cinerea*.
Therefore, we can prove that PolycoBead 2.0 as our final
product can maintain the activity of the effective constituent
during storage, show good results in application, and bring
more benefits and convenience to farmers when used.

* [ ]
  References
  1. [1] FAO, IFAD, UNICEF, WFP and WHO. 2023. The State of Food
     Security and Nutrition in the World 2023. Urbanization,
     agrifood systems transformation and healthy diets across the
     rural-urban continuum. Rome, FAO.
     [2] The World Bank. Agriculture and food .[cited 2023-09-15].
     https://www.worldbank.org/en/topic/agriculture/overview#1
     [3] FAO. Crops and livestock products.[cited 2023-09-15].
     https://www.fao.org/faostat/en/#data/QCL/visualize
     [4] Williamson B, Tudzynski B, Tudzynski P, van Kan JA.
     Botrytis cinerea: the cause of grey mould disease. Mol Plant
     Pathol. 2007 Sep;8(5):561-80.
     [5] FAO. Prevention of post-harvest food losses fruits,
     vegetables and root crops a training manual. Rome, 1989.
     https://www.fao.org/3/T0073E/T0073E02.htm
     [6]Rupp S, Plesken C, Rumsey S, Dowling M, Schnabel G, Weber
     RWS, Hahn M. Botrytis fragariae, a New Species Causing Gray
     Mold on Strawberries, Shows High Frequencies of Specific and
     Efflux-Based Fungicide Resistance. Appl Environ Microbiol.
     2017 Apr 17;83(9) :e00269-17.
     [7]
     叶艳英,熊春晖,袁经相等.植物源杀菌剂防治灰霉病研究进展[J].生物灾害科学,2016,39(01):27-31.
     [8]
     杜颖,付丹妮,邹益泽等.2017年辽宁省番茄灰霉病菌对腐霉利的抗药性现状及机制研究[J].中国蔬菜,2018(01):58-65.
     [9] Agroinfo. The latest survey of biological pesticide
     registration in China. G Sun, 2019.
     [10] Chinapesticide. China pesticide registration data.
     http://www.chinapesticide.org.cn/zwb/dataCenter

{% endblock %}

{% extends "layout.html" %} {% block title %}Experiments{% endblock %} {% block
lead %}Describe the research, experiments, and protocols you used in your iGEM
project.{% endblock %} {% block page\_content %}

## Experiments

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# Menu

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[Overview](#anchor1)

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[Timelog](#anchor2)

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setting

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* [ ]
  References
  1. [1]. Lung Cancer Statistics | How Common is Lung Cancer?
     (n.d.). Retrieved October 5, 2022, from
     https://www.cancer.org/cancer/lung-cancer/about/key-statistics.html
     [2]. Poulet, G., Massias, J., & Taly, V. (2019). Liquid
     Biopsy: General Concepts. Acta Cytologica, 63(6), 449–455.
     https://doi.org/10.1159/000499337
     [3]. Strimbu, K., & Tavel, J. A. (2010, November). What are
     biomarkers? Current Opinion in HIV and AIDS, 5(6), 463–466.
     https://doi.org/10.1097/coh.0b013e32833ed177
     [4]. Kristensen, L. S., Andersen, M. S., Stagsted, L. V. W.,
     Ebbesen, K. K., Hansen, T. B., & Kjems, J. (2019, August 8).
     The biogenesis, biology and characterization of circular RNAs.
     Nature Reviews Genetics, 20(11), 675–691.
     https://doi.org/10.1038/s41576-019-0158-7
     [5]. Chen, L., & Shan, G. (2021, May). CircRNA in cancer:
     Fundamental mechanism and clinical potential. Cancer Letters,
     505, 49–57. https://doi.org/10.1016/j.canlet.2021.02.004
     [6]. Di, X., Jin, X., Li, R., Zhao, M., & Wang, K. (2019,
     March). CircRNAs and lung cancer: Biomarkers and master
     regulators. Life Sciences, 220, 177–185.
     https://doi.org/10.1016/j.lfs.2019.01.055
     [7]. Wang, C., Tan, S., Li, J., Liu, W. R., Peng, Y., & Li, W.
     (2020, November). CircRNAs in lung cancer - Biogenesis,
     function and clinical implication. Cancer Letters, 492,
     106–115. https://doi.org/10.1016/j.canlet.2020.08.013
     [8]. Wang, S., Zhang, K., Tan, S., Xin, J., Yuan, Q., Xu, H.,
     Xu, X., Liang, Q., Christiani, D. C., Wang, M., Liu, L., & Du,
     M. (2021, January 11). Circular RNAs in body fluids as cancer
     biomarkers: the new frontier of liquid biopsies. Molecular
     Cancer, 20(1). https://doi.org/10.1186/s12943-020-01298-z
     [9]. DNA Nanotechnology Tools: From Design to Applications.
     (2022, April 22). Wyss Institute. Retrieved October 5, 2022,
     from
     https://wyss.harvard.edu/technology/dna-nanotechnology-tools-from-design-to-applications/
     [10]. Jiao, J., Xiang, Y., Duan, C., Liu, Y., Li, C., & Li, G.
     (2020, August 24). Lighting Up CircRNA Using a Linear DNA
     Nanostructure. Analytical Chemistry, 92(18), 12394–12399.
     https://doi.org/10.1021/acs.analchem.0c02146

{% endblock %}

{% extends "layout.html" %} {% block page\_content %}

## Results

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# Menu

[ ]

[Overview](#anchor1)

[ ]

[RNAi therapy](#anchor2)

[shRNA stage](#anchor2.1)

[CPP+shRNA stage](#anchor2.2)

[ ]

[Plant immunity](#anchor3)

[Construction and transformation of plasmids](#anchor3.1)

[Expression of Proteins](#anchor3.2)

[DAB staining experiment](#anchor3.3)

[References](#refer)

## Overview

In our project, we have combined RNAi and plant immunity to
control tomato gray mold disease.

**In the experiments of RNAi, we obtained the following
results:**

* -We designed shRNA and ligated it into the pET-28a(+) plasmid.
  The target shRNA was expressed through induction in
  *Escherichia coli* HT115(DE3).

* -We combined cell-penetrating peptide (CPP) BP100-(KH)9
  with shRNA and observed their morphological structure using
  scanning electron microscopy (SEM).

* -We applied naked shRNA, BP100-(KH)9+shRNA, and
  BP100-(KH)9+bi-shRNA to tomato fruits, and
  demonstrated the effectiveness of our RNAi by calculating the
  distribution of disease spots.

* -We applied naked shRNA, BP100-(KH)9+shRNA, and
  BP100-(KH)9+bi-shRNA to tomato fruits, and
  demonstrated the good silencing effect of our shRNA on the
  target genes through qRT-PCR.

**In the experiments of plant immunity, we conducted the
following three aspects of work:**

* -We constructed the BvEP plasmid for *E. coli* BL21(DE3)
  and *B. subtilis* WB800. And we verified the successful
  transformation.

* -We obtained the optimal induction conditions for BvEP and
  verified its successful expression in BL21(DE3).

* -We validated BvEP's role in triggering plant immunity and
  demonstrated that the intensity of the immune response increased
  as protein concentration increased.

## RNAi therapy

### shRNA stage

When deciding to use RNAi technology to kill
*Botrytis cinerea*, we need to consider which small RNA
(sRNA) molecules can trigger the RNAi process. We first noticed
that siRNA molecules can enter the cells and load on RISC to
effect. Knowing the instability of siRNA and considering
production costs, we decided to design shRNA to silence the
target gene. This way, we can use *E. coli* in the
laboratory for low-cost but mass production of shRNA.

shRNA can be recognized and processed into siRNA by Dicer, and
then combines with Ago protein to form the RNAi silencing
complex (RISC). RISC, consisting of Argonaute-2, Dicer, and TRBP
proteins, can then specifically silence the expression of target
genes.

Our RNAi method is Spray-Induced Gene Silencing (SIGS), which
has been widely used in agricultural research since its first
application in 2016. It is a new non-transformation strategy
that suppresses pathogenic gene expression by spraying RNAi
molecules produced exogenously onto plant tissues. The sprayed
RNAi molecules are absorbed by cells through stomata or
lenticels, and move throughout the body via hyphae or phloem
sieve tubes, causing gene silencing and induced mortality
without transformation.

#### Selection of shRNA Molecular Target Genes

We have selected a total of 6 target genes as our RNAi silencing
targets. The information of these target genes is shown in the
Table 1. *BcchsIIIa* and *Bccyp51* are essential genes
for the survival of *B. cinerea*, while *BcOAH1*,
*Bcpme1*, *Bcdcl1*, and *Bcdcl2* are key genes
involved in the infection process of *B. cinerea* on
tomatoes. Furthermore, we have concatenated 2 shRNAs targeting
the survival genes of *B. cinerea* with the specific
sequence ATGCCT, to build a bi-shRNA named shRNA(Box-survival).
We have also stringed 4 shRNAs targeting the key genes involved
in *B. cinerea* infection on tomatoes, and named it
shRNA(Box-infect).

**Table 1. Target Genes and their Target Sequences of
shRNA**

![](https://static.igem.wiki/teams/4653/wiki/results/szu-result-gene.png)

After selecting the above target genes, we designed the
corresponding shRNA molecule through the CDS sequence of the
target gene. After BLAST and confirmation of its specificity in
the total nucleic acid library, we assembled it through the
sequence of XbaI restriction site - sense RNAi fragment - loop -
reverse antisense RNAi fragment - BlpI restriction site, and
connected it to the pET-28a (+) plasmid to obtain our shRNA
expression vectors.

#### IPTG induced production

After plasmid extraction, we transformed the constructed shRNA
expression vector into *E. coli* HT115(DE3) and performed
PCR. Our specific primers successfully amplified a 260 bp band
from the plasmid, confirming the successful transformation of
the plasmid (Figure 1). This indicates that the shRNA expression
vector has been successfully introduced into *E. coli* and
can be detected and confirmed by PCR. This is an important
milestone that lays the foundation for further experiments.

![](https://static.igem.wiki/teams/4653/wiki/results/szu-engineering-plasmid-2.png)

**Figure 1. Agarose Gel Electrophoresis of Plasmids after
Plasmid PCR
1-5: Plasmids control; 5-10: Plasmids extracted from
*E. coli*.**

After induction with IPTG, the RNA was extracted using the
Trizol method and the results shown in Figure 2 were obtained
after electrophoresis. Compared to the non-induced sample, there
is a brighter band between 50-100 bp and 100-150 bp in the
induced sample lane. Our shRNAs have the size of 69 bp, while
the Box-survival, which is a concatenation of two shRNAs, have
the size of 124 bp. This confirms the successful extraction of
our shRNA, and the generated shRNA is of the expected size.

![](https://static.igem.wiki/teams/4653/wiki/results/szu-results-rnai-2.jpg)

**Figure 2. Electrophoresis of RNA extracted from
*E. coli* HT115 (DE3)**

## CPP+shRNA Stage

However, the instability of shRNA in the field environment
hinders the optimal performance of our product. Understanding
our expectations, our PI suggested that we could try using
cell-penetrating peptides (CPP) in combination with shRNA for
spray application, and provided us with BP100-(KH)9
as our CPP material. BP100-(KH)9 is a carrier
peptide-based gene delivery system that enhances the endocytic
uptake and cytoplasmic transfer of shRNA in plants, allowing for
more efficient transfection of plant callus cells with shRNA. To
understand the morphology of the shRNA and BP100-(KH)9
complex, we used scanning electron microscopy (SEM) to observe
the morphology of shRNA, BP100-(KH)9, and
shRNA+BP100-(KH)9 separately. As shown in the Figure
3, we observed that shRNA and BP100-(KH)9 form
spherical aggregates under electron microscopy. These small
spherical aggregates further tend to aggregate with each other.
We speculate that this stacking aggregation is due to
electrostatic forces.

![](https://static.igem.wiki/teams/4653/wiki/results/szu-results-rnai-3.png)

**Figure 3. Morphology of shRNA+CPP under Scanning Electron
Microscope at 40,000x Magnification**

**More information on the RNAi process, including phenotype
statistics and molecular validation, can be found in
[Proof of Concept](https://2023.igem.wiki/szu-china/proof-of-concept)**

## Plant Immunity

In the early stage of the plant immune induction project, we
selected several potential inducers of plant immune response based
on their characteristics for proof-of-concept. One such
immune-inducing factor is oligogalturonic acid (OGs), which
belongs to the damage-associated molecular patterns of plant
cells. It is a kind of oligosaccharide produced after the
destruction of plant cell wall component pectin by pathogen
infection. It can be recognized and bound by pattern recognition
receptors (PRRs), thereby stimulating downstream immune pathways.
We used PehA, a polygalacturonate endonuclease, as our
immune-inducer to induce plant immunity by targeting the plant
cell wall to produce OGs.

The other is induced factors which are Pathogen-associated
molecular patterns. In this study, we selected Pseudomonas
aeruginosa flagellin, which has a 22-amino acid N-terminal peptide
that can activate downstream immune pathways by binding to the
cell surface receptor FLS2-BAK1. This peptide flg22 was used as an
immune-inducing factor, and three flg22 were concatenated to
obtain 3x flg22 to verify their immune effects.

We also hope to stimulate the ETI response in plants, so that
plant cells can produce a stronger and sustained immune effect. We
selected BvEP, a protein phosphomutase from
*Bacillus velezensis* LJ02, in the hope that its expression
in *Bacillus subtilis* would activate tomato sustained
defense.

### Construction and transformation of plasmids

To verify whether the selected immune-inducing factors PehA,
Flg22, 3xFlg22 and BvEP could activate plant immunity, we
constructed their gene expression vectors in *E. coli* and
all of their expression were regulated by IPTG.

![](https://static.igem.wiki/teams/4653/wiki/results/pathway-1.png)

**Figure 4. Gene pathways for immune-inducing factor
expression.**

After constructing the expression vectors of the immune-inducing
factors above, we transferred these recombinant plasmids into
*E. coli* BL21 (DE3), and then screened positive monoclonal
colonies in resistant plates for expansion culture and plasmid
extraction. The cloned products containing the target fragment
were amplified with specific primers and verified by DNA agarose
gel electrophoresis. As shown in Figure 5, electrophoresis
results showed that the bands of different recombinant plasmids
amplified by PCR were all of the expected sizes, indicating that
our plasmids were successfully transformed into
*E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-4.jpg)

**Figure 5. Results of amplification of different recombinant
plasmids by specific primers.**

**(a)PCR of pGS21a-PehA. M: DL2000 DNA marker; 1: PehA.
(b)PCR of 1x Flg22, 3x Flg22, Flagellin full length and BvEP
in vector pET-28a. M: DL2000 DNA marker; 1-4: 1x Flg22, 3x
Flg22, Flagellin full length and BvEP.**

### Expression of Proteins

The expression of immune protein factors is regulated by the lac
operon, so IPTG was applied to induce our protein expression.
The PehA proteins were extracted using the GST-tag protein
purification kit, and the rest of the proteins were extracted
using the His-tag protein purification kit. After extraction of
the proteins we induced to express, the protein concentration
was determined by the BCA assay. BvEP was expressed at very low
levels under conventional induction conditions. We then
optimized this assay and found that protein expression levels
were higher when induced at 100 rpm in a shaker at 20 ° C. The
protein concentration of BvEP in 4.5 mL volume of induction
culture was measured at 4 h, 8 h, and 16 h of induction, and the
expression concentration of BVEP protein was the highest after
16h of induction, reaching 0.530 mg/ml.

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-5.jpg)

**Figure 6. BCA standard curve.**

In addition, we verified the extracted target proteins by
SDS-PAGE after 16 h of induction. As shown in the figure, Figure
7a shows the BvEP protein electrophoresis after 16h of IPTG
induction. By comparing with the total protein of *E. coli*
BL21(DE3) without induction, it can be seen that there is a
clear band between 14.4kD and 4kD. The predicted size of BvEP
protein was 12.1kD, indicating that this band was our target
protein. Similarly, in Figure 7b, an obvious band between 35kD
and 25kD appeared after 16 h of IPTG induction compared with the
non-induced sample, while the size of our Flagellin full length
protein was 30.6kD, showing that it was successfully induced.

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-6.jpg)

**Figure 7. The induced expression of target proteins
detected by SDS-PAGE.**

**(a)Induced expression of BvEP. (b)Induced expression of
Flagellin full length.**

In addition, our target proteins were also verified in Western
Blot experiments. As can be seen from Figure 8, there is a
specific band between 15kD and 10kD, and the protein size of
BvEP is 12.1kD, indicating that the protein extracted from the
induced *E. coli* is indeed our target protein.

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-7.jpg)

**Figure 8 Western Blot validation of BvEP induced
expression**

However, when we tried to express PehA, 1x flg22, and 3x flg22,
the amount of the proposed protein was found to be too low for
subsequent DAB experiments and was therefore abandoned in later
experiments.

### DAB staining experiment

The burst of ROS is a typical response to the activation of
plant immunity, so we performed DAB staining experiments to
examine the degree of ROS burst in tomato leaves after treatment
with different concentrations of BvEP. The darker the DAB
staining results, the more ROS burst in the leaves. In that
experiment, our control group 1 was treated with clean water and
control group 2 was treated with total protein extracted in
*E. coli* BL21(DE3) without induction (Figure. 9).

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-8.jpg)

**Figure 9. DAB staining to detect the intensity of immunity
triggered by different concentrations of BvEP.**

**(a)Soaking method to apply BvEP. Low: 10ug/ul, Mid:
25ug/ml, High: 50ug/ml. (b)Dropping method to apply BvEP.
Low: 40ug, Mid: 100ug, High: 200ug.**

After staining, ImageJ software was used to perform gray scale
analysis of the leaves treated with the two different BvEP
methods to quantify the immune intensity, followed by t-test to
determine the significant difference between the different BvEP
concentrations. Lower gray levels, that is, more brown
precipitates in leaves, indicate a stronger burst of ROS. Each
treatment contains a sample number of 9, and the resulting data
are shown in Figure 10.

![](https://static.igem.wiki/teams/4653/wiki/parts/szu-parts-k4653105-9.jpg)

**Figure 10. Gray levels in different leaves measured by
ImageJ software.**

**(a)The gray value of leaves treated protein after soaking.
(b)The gray value of leaves after dropping. (ns P > 0.05; \*
P < 0.05; \*\* P < 0.01; \*\*\* P < 0.001; \*\*\*\* P < 0.0001).**

As can be seen from the figure above, the degree of ROS burst in
leaves increases with the concentration of applied BvEP, whether
using the soaking or dropping method. Through the comparison of
the two methods, we can obviously observe that the ROS burst in
the leaves treated by soaking method is more than that by
dropping method.

More details can be viewed in
[Proof of concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D).

* [ ]
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