iGEM 2023 — Team hnu-china

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![img](https://static.igem.wiki/teams/4752/wiki/notebook-title1.png)

## 30

### Sep

## 2022

## November

### November 10th

Brainstorming

## December

### December 1st

Check the project and the members

## 2023

## March

### March 10th

Finish our exams and be ready to start our experiments.

## April

### March 30 to April 13

Construction of the NCED3-RUBY system

Click **[here](https://static.igem.wiki/teams/4752/wiki/3-30-4-13.pdf)** to see our notebook.

## May

### May 19th

Large-scale extraction of the NCED3-RUBY plasmid.

Click **[here](https://static.igem.wiki/teams/4752/wiki/5-19.pdf)** to see our notebook.

## June

### June 22 to June 28

PCR amplification of GLR2.9, enzyme digestion of the NCED3-RUBY plasmid, CEII ligation, verification of PCR products, and investigation of the reasons for band diffusion. Transformation of DH5α and culturing of colonies on plates.

Click **[here](https://static.igem.wiki/teams/4752/wiki/6-22-6-28.pdf)** to see our notebook.

## July

### July 7 to July 15

Meeting with human practice supervisors: Brainstorming, integration to the team.

Click **[here](https://static.igem.wiki/teams/4752/wiki/7-1-7-15.pdf)** to see our notebook.

### July 16 to July 31

Receipt of NCED3-RUBY plasmid seeds, initiation of plant experiments, preparation of 1/2 MS solid medium, initial planting trials, T4 ligation, extraction of the basta plasmid, enzyme digestion, and bacterial shaking. Re-amplification of GLR2.9, extraction of BX fragments, and shaking with Agrobacterium.

Click **[here](https://static.igem.wiki/teams/4752/wiki/7-16-7-31.pdf)** to see our notebook.

## Augest

### August 1 to August 15

Plant experiments including tobacco transplantation, Agrobacterium transformation into tobacco leaves, and drought experiments. Molecular experiments including validation of GLR2.9 PCR systems, construction of GLR2.9-RUBY systems, and colony PCR validation for E. coli and Agrobacterium.

Click **[here](https://static.igem.wiki/teams/4752/wiki/8-1-8-15.pdf)** to see our notebook.

### August 16 to August 30

Plant experiments involving the planting of NCED3-RUBY seeds for drought validation and tobacco transplantation. Molecular experiments including sequence validation by PCR, enzyme digestion, ligation to construct NCED3-Bx, preparation of permeation buffer, E. coli transformation, shaking, and plasmid extraction for NCED-Bx validation, as well as GLR2.9-RUBY-related activities.

Click **[here](https://static.igem.wiki/teams/4752/wiki/8-16-8-30.pdf)** to see our notebook.

## September

### September 1 to September 27

Molecular Experiments:Agrobacterium Transformation and Verification in Various Systems
Plant Experiments: Agrobacterium infiltration, leaf injection, simulation of Drought Experiment, time-lapse Photography.

Click **[here](https://static.igem.wiki/teams/4752/wiki/9-1-9-13.pdf)** to see our notebook.

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![img](https://static.igem.wiki/teams/4752/wiki/engining.jpg)

- [**Project Design**](#eng_1)

* [**Response to Drought**](#eng_2)
* [**Response to Biological Stress**](#eng_3)
* [**Plant Experiments**](#eng_4)

- [**Experiment Planning**](#eng_5)

## Project Design

---

After reading the literature extensively, we decided to build two reporting systems to warn of drought stress and pest stress.

### 1.Early Warning System in Response to Drought:NCED3-RUBY

Since drought can induce the expression of CLE25 in the root, and can be transferred from the root to the leaf for a long distance to bind to the receptor proteins BAM1 and BAM3, it induces NCED3 expression, resulting in an increase in ABA concentration and a decrease in the degree of stomatal openness in response to plant water shortage. Therefore, we chose the promoter of NCED3, one of the key enzymes in ABA biosynthesis, in response to drought stress.

![img](https://static.igem.wiki/teams/4752/wiki/enger1.jpg)

Meanwhile, the Department of Cell and Developmental Biology at the University of California, San Diego and the State Key Laboratory of Crop Genetics and Germplasm Improvement at Nanjing Agricultural University published a team entitled "A reporter for noninvasively monitoring gene expression and plant transformation" in Horticulture Research on September 19, 2020 A research-oriented article that focuses on a betanoside-based chromogenic reporting system called RUBY. The system turns the plant red without the help of any instruments, and can be observed with the naked eye alone, which is a good visualization system, so we chose RUBY as our reporting system.

In the team's expected results, the plants that received the NCED3promoter-RUBY early warning system would appear red visible to the naked eye under drought stress.

![img](https://static.igem.wiki/teams/4752/wiki/enger2.png)

---

### 2.Early Warning System in Response to Biological Stress: GLR2.9-Bx

At present, the immune mechanism of plant bodies in response to pest and disease stress is not completely clear, and after reviewing the literature, it is found that glutamic acid, as a signaling substance for plant mechanical damage, increases the concentration of calcium ions in the cytoplasm and conducts to other parts of plants by being perceived by the glutamate receptor-like protein family, thereby activating the systematic defense response of plants in advance. Further studies found that GLR2.9 was involved in the innate immune response of Arabidopsis thaliana and was significantly upregulated during bacterial infection, which was suitable as a receptor in response to biological stress.

In the literature research, it was found that Professor Xiong Aisheng's team from the State Key Discipline of Vegetable Science/State Key Laboratory of Crop Genetics and Germplasm Innovation of Nanjing Agricultural University published a research brief entitled "A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation" online in The Crop Journal. Using the visual color and fluorescence characteristics of betaxanthin, a convenient reporting system Bx was developed and designed, which turned the plants yellow and produced bright green fluorescence under the excitation of blue light.

![img](https://static.igem.wiki/teams/4752/wiki/enger3.png)

---

### 3. Plant Experiments

After designing the two reporting systems, the team intends to cross-combine the promoter and the reporting system, so as to not only obtain a variety of early warning systems, but also make it easier to verify the success of the early warning system. As our first successful system, we decided to use NCED3promoter-RUBY to perform stable transfer treatment on Arabidopsis thaliana plants to obtain stable traits to observe plants. The rest of the system uses teleportation technology to observe the phenomenon on tobacco.

## Experiment Planning

---

It can be seen that our experiment is divided into two parts: molecular experiment and plant experiment, and two reporting systems are divided into two reporting systems and carried out in parallel.

So in the molecular experiment part, the team was divided into two groups, one group was responsible for the construction of the NCED3promoter-RUBY early warning system, and the other group was responsible for the construction of the GLR2.9 promoter-Bx early warning system.

The molecular experimental part starts with the most basic plasmid construction, and the NCED3/GLR2.9 promoter fragment is obtained in the Arabidopsis whole DNA set, and ligated with the target gene fragment synthesized from the biocompany to construct a complete plasmid.

After obtaining the plasmid, it was transformed into E. coli and compared with the sequencing results to confirm that the target gene and promoter gene were intact.

After the plasmid is determined to be correct, agrobacterium transformation is performed. Among them, NCED3promoter-RUBY was stabilized to obtain stable plants. The rest of the early warning system is instantaneous on the tobacco to see if the reporting system is achieving the desired effect.

After verifying that the early warning system can achieve the expected effect, the transgenic plants are transformed to obtain the final product of the project.

### Reference

Sato H, Takasaki H, Takahashi F, et al. Arabidopsis thaliana NGATHA1 transcription factor induces ABA biosynthesis by activating NCED3 gene during dehydration stress[J]. Proceedings of the National Academy of Sciences, 2018, 115(47): E11178-E11187.

He Y, Zhang T, Sun H, et al. A reporter for noninvasively monitoring gene expression and plant transformation[J]. Horticulture research, 2020, 7.

Köster P, DeFalco T A, Zipfel C. Ca2+ signals in plant immunity[J]. The EMBO journal, 2022, 41(12): e110741.

Deng Y, Duan A, Li T, et al. A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation[J]. The Crop Journal, 2023, 11(2): 666-671.

Yuanjie Deng , Aoqi Duan , Tong Li, Huiru Wang, Aisheng Xiong.A betaxanthin-based visible and fluorescent reporter for monitoring plant transformation The Crop Journal 11 (2023) 666–671.

Yubing He , Tao Zhang, Hui Sun, Huadong Zhan and Yunde Zhao. A reporter for noninvasively monitoring gene expression and plant transformation Horticulture Research (2020) 7:152.

Schwartz, S H et al. “Specific oxidative cleavage of carotenoids by VP14 of maize.” Science (New York, N.Y.) vol. 276,5320 (1997): 1872-4.

BABAK Behnam et al. “Characterization of the Promoter Region of an Arabidopsis Gene for 9-cis-Epoxycarotenoid Dioxygenase Involved in Dehydration-Inducible Transcription” DNA RESEARCH 20,(2013) 315–324.

Strack, D., Vogt, T. & Schliemann, W. Recent advances in betalain research. Phytochemistry 62,247–269 (2003).

Xu, J.-J., Fang, X., Li, C.-Y., Yang, L. & Chen, X.-Y. General and specialized tyrosine metabolism pathways in plants. aBIOTECH, https://doi.org/10.1007/s42994019-00006-w (2019).

Polturak, G. & Aharoni, A. Advances and future directions in betalain metabolic engineering. N. Phytol. 224,1472–1478 (2019).

Gandía-Herrero, J. Escribano, F. García-Carmona, Betaxanthins as pigments responsible for visible fluorescence in flowers, Planta 222 (2005) 586–593.

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![img](https://static.igem.wiki/teams/4752/wiki/experiments.jpg)

- [**Introduction**](#exp_1)
- [**NCED3-RUBY Drought Warning System**](#exp_2)
- [**GLR2.9-Bx Biological Stress Warning System**](#exp_3)

## Introduction

---

Genetic modification of plants is a complex and time-consuming process, but in order to expedite the validation of our plant warning system, we have devised the following comprehensive experimental protocol

We have employed the components listed in the table below to assemble our plant warning system:

| System Name | Promoter | Reporter System | Critical Elements |
| --- | --- | --- | --- |
| Drought Warning System | NCED3 promoter | RUBY reporter | CYP76AD1, DODA, GT (glucosyltransferase) |
| Biological Stress Warning System | GLR2.9 promoter | Bx reporter | BvCYP76AD6, BvDODA1 |

## NCED3-RUBY Drought Warning System

---

## Extraction Genes

## Construction Vector

## Agrobacterium Transformation

## Transient Transformation

## Stable Transformation

## Extraction of NCED3 Promoter

In this step, we identified the sequence of the NCED3 gene through the analysis of the plant genome. We selected a 2000bp region upstream of the ATG start codon as the NCED3 promoter sequence. Subsequently, we designed homologous recombination primers based on the SmaI-PstI restriction enzyme sites and plasmid fragments to amplify the NCED3 promoter. PCR reactions were performed, resulting in the generation of DNA fragments containing the NCED3 promoter with homologous arms.

![img](https://static.igem.wiki/teams/4752/wiki/frame-61.png)

## Construction of Pro:NCED3-RUBY Expression Vector

In this step, the pDR5-RUBY plasmid was subjected to SmaI-PstI double digestion to obtain digested plasmid fragments. The NCED3 promoter was then ligated with the digested plasmid to create an expression vector harboring the NCED3 promoter. These plasmids were subsequently transformed into DH5α Escherichia coli and plated on LB agar supplemented with kanamycin for selection and cultivation. Single colonies were picked from the plates for PCR and agarose gel electrophoresis verification, followed by sequencing to ensure the correct construction of the plasmid.

![img](https://static.igem.wiki/teams/4752/wiki/frame-62.png)

## Agrobacterium Transformation

In this step, validated single colonies were subjected to oscillation culture to amplify and extract the target vector plasmids. These target plasmids were then transformed into Agrobacterium GV3 101 in a competent state. The transformed Agrobacterium was plated on LB agar plates containing rifampicin and kanamycin for selection and cultivation. Single colonies were picked from the plates for PCR and agarose gel electrophoresis verification to confirm the successful transformation of the plasmid into Agrobacterium.

## Transient Transformation and Induction of Expression in Tobacco

In this step, Agrobacterium cultures containing the target gene plasmids were oscillated, and the cells were centrifuged and resuspended to prepare a suspension. The suspension was introduced into tobacco leaves through injection. Both injected and non-injected leaves from the same tobacco plant were maintained to create conditions for drought stress and non-drought stress. Subsequently, time-lapse photography was conducted to observe whether leaf color changes occurred under drought conditions, thereby validating the effectiveness of the system.

![img](https://static.igem.wiki/teams/4752/wiki/frame-67.png)

## Stable Transformation Verification in Arabidopsis

### 1.Rapid Verification

In this step, the extracted target plasmids were sent to a company for stable transformation. The transformed Arabidopsis seeds with the warning system and wild-type Arabidopsis seeds were subjected to drought stress on 1/2MS agar plates. The growth status and leaf color of the seeds were observed to validate the system's effectiveness.

![img](https://static.igem.wiki/teams/4752/wiki/frame-71.png)

### 2.Formal Experiment

Transplanting seedlings into soil pots with consistent initial weights, a stable cultivation period of three weeks was allowed. Subsequently, a drought stress experiment was initiated, simulating natural drought conditions based on the duration of withholding water to emulate varying degrees of drought severity. Leaf color changes were observed and assessed during this drought stress experiment.

![img](https://static.igem.wiki/teams/4752/wiki/frame-68.png)
![img](https://static.igem.wiki/teams/4752/wiki/frame-69.png)

## GLR2.9-Bx Biological Stress Warning System

---

## Extraction Genes

## Construction Vector

## Agrobacterium Transformation

## Transient Transformation

## Extraction of GLR2.9 Promoter and Synthesis of Bx Gene

Through the analysis of the plant genome, we identified the sequence of the GLR2.9 gene. To improve the success rate of subsequent steps involving multi-fragment assembly, we selected the 1500bp sequence upstream of the ATG start codon as the GLR2.9 promoter. As we did not have the sequence of the Bx gene, we entrusted a company to synthesize it. Subsequently, we designed homologous recombination primers to amplify the GLR2.9 promoter and the Bx reporter gene sequences, using the linearized vector fragment after NcoI-PstI digestion. PCR reactions were conducted to obtain GLR2.9 promoter DNA fragments and Bx reporter gene fragments, both containing homologous arms.

![img](https://static.igem.wiki/teams/4752/wiki/frame-63.png)

## Construction of Pro:GLR2.9-Bx Expression Vector

We performed NcoI-PstI double digestion on the pCAMBIA1300-C-MCS-GFP plasmid to obtain a linearized vector fragment. Next, we carried out homologous recombination by ligating the GLR2.9 promoter DNA fragment, Bx reporter gene fragment, and the linearized vector fragment, resulting in the expression vector plasmid containing the GLR2.9-Bx reporter system. These plasmids were then transformed into DH5α Escherichia coli and spread on LB agar plates supplemented with kanamycin for selection. Single colonies were picked from the plates and subjected to PCR and agarose gel electrophoresis verification, followed by sequencing to ensure correct plasmid construction.

![img](https://static.igem.wiki/teams/4752/wiki/frame-64.png)

## Agrobacterium Transformation

The verified single colonies were cultured with agitation, and the target vector plasmids were extracted. These target vector plasmids were subsequently transformed into GV3 101 Agrobacterium tumefaciens in a state of competence. The transformed Agrobacterium cultures were spread on LB agar plates containing rifampicin and kanamycin for selection. Finally, single colonies were selected from the plates and subjected to PCR and agarose gel electrophoresis verification to confirm the successful transformation of the plasmid into Agrobacterium.

## Transient Transformation and Induction of Expression in Tobacco

The Agrobacterium cultures containing the target gene plasmids were agitated, and the cells were pelleted and resuspended to prepare a suspension. This suspension was injected into tobacco leaves using a syringe. On the same tobacco plant, both injected and non-injected leaves were present. To simulate biological stress, Pst DC3000 bacteria, commonly used in the laboratory for plant biotic stress studies, were used to treat the leaves, while the control group leaves were sprayed with water. Subsequently, time-lapse photography was conducted to observe any color changes in the leaves, confirming the system's effectiveness. Furthermore, based on the property of Bx emitting green fluorescence under blue light, treated leaf samples were collected for fluorescence microscopy to further validate the results.

![img](https://static.igem.wiki/teams/4752/wiki/frame-70.png)
![img](https://static.igem.wiki/teams/4752/wiki/frame-69.png)

Click here to access the experiment's [Protocol](https://2023.igem.wiki/hnu-china/protocol)!

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