iGEM 2023 — Team csu-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block image %}

![](https://static.igem.wiki/teams/4585/wiki/notebook-00.png)

{% endblock %}
{% block content %}

# FEBRUARY

February 28th  Built our team ,  work and begin fundraising

February 1 to February 28  Brainstorm

Read the competition requirements.
Studied former projects and their advantages, leared and studied synthetic biological content.Design our project.
Determine team goals and tasks.

# April

April 2nd  Conducting debates

In order to explore the public's understanding and awareness of rare diseases, a debate on traditional and new therapies was organized. Students from various disciplines made different suggestions for our project to ensure that the follow-up project is more relevant.

April 16th  Visit pharmaceutical companies

Our members visited Changsha Jingyi Pharmaceutical Technology Co., Ltd. to learn about the long process of new drugs from practice to clinic, and to provide new ideas for our experiments from theory to clinic.

April 21st  Interview with Doctor

We interviewed Dr. Meichao Men in order to understand the knowledge and current technological bottlenecks related to diseases caused by abnormal GnRH secretion, to provide a theoretical basis for our cell-embedded therapy and to find the optimal solution for treatment.

# June

June 4th  Exchange with Nanjing-China team

We had an online Tencent meeting with the Nanjing-China team, which provided new inspiration for both teams and useful advice in the process of learning from each other.

June 11th  Exchange with XMU-CHINA team

We had an online Tencent meeting with the XMU-CHINA team, which promoted mutual understanding between the two universities and discussed matters related to the award submission.

June 26th  Distributed questionnaires to patients with Kalman Syndrome

We distributed and collected questionnaires for people with Kalman Syndrome to understand the main problems faced by people with Kalman Syndrome.
Design wiki
We interviewed Dr. Meichao Men in order to understand the knowledge and current technological bottlenecks related to diseases caused by abnormal GnRH secretion, to provide a theoretical basis for our cell-embedded therapy and to find the optimal solution for treatment.

# August

August 8th  Northwest Networking Meeting

We held a joint exchange with 6 other teams to promote communication and collaboration between schools.

August 13th  Exchange with NWU-CHINA team

We conducted an online Tencent meeting with the NWU-CHINA team, and the two teams discussed further about the activities of the HP group to try more popular social media.

August 14th  Exchange with WHU-CHINA team

We had an online Tencent meeting with the WHU-CHINA team to expand each other's careers in research projects and explore more innovative breakthroughs.

August 19th  Participation in Rare Disease Alumni Meeting

We participated in the Kallman Syndrome Alumni Meeting as a staff member to collect their views on existing and novel therapies, which provided us with optimization ideas for the engineered cells we designed.

# OCTOBER

Improved the details of our wiki

October 26th-October 28th  Giant Jamboree

Program review

# March

March 1th  Determined the theme.

Analyzed former awards, made sure criteria for special prizes and medals.
Designed basic structure, arranged our works every month during the competition.

# May

May 13th  Interview with Kalman patient

In order to know the real needs of patients, we interviewed Kalman Syndrome patients on a one-to-one basis, which pointed out the direction for the research and publicity of our project.

May 26th  Interview with Kalman Syndrome Patients' Association - Old K Home

We had an online conversation with the head of the association to learn more about the charity's journey and gained useful guidance from them for our project.

May 28th  Communication with HNU-CHINA team

We had an online Tencent meeting with the HNU-CHINA team, which deepened the understanding of each other's projects between the two schools and facilitated new inspiration for the iGEM team.

# July

July 15th  Yuanling No.1 Middle School Practice Science and Technology

Our team members followed the teaching team from Xiangya School of Medicine of Central South University to teach in Yuanling No.1 Middle School, which improved the students' understanding of synthetic biology and cultivated their passion for life science.

July 18th  Lanshan No.2 Middle School Practicing Science

We followed the teaching team from the School of Life Sciences of Central South University (CSU) to Lanshan County, Hunan Province, to help children in the mountainous areas to learn objective scientific knowledge, and to call on the children to see rare diseases, understand rare diseases, and respect rare diseases.

July 19th  Communication with LZU-CHINA team

We conducted an online Tencent meeting with the LZU-CHINA team to promote the clash of ideas between the two schools and further exploration of the medical field by the teams from both schools.

July 22nd  joint presentation by each school

We worked closely with LZU-CHINA to conduct summer teaching activities in local elementary schools to popularize synthetic biology knowledge.

July 28th  Distribute questionnaires on the public's understanding of rare diseases

We distributed and collected questionnaires on the general public's understanding of GnRH, so that our ideas could be more relevant to the public's needs.

# September

September 3rd  iG20 Video Promotion

We joined hands with other teams to make a popularization video of synthetic biology, hoping to draw more attention to it.

September 11th  iGEM competition for freshmen students

We seized the school season to introduce our project to let new students better understand synthetic biology and enhance their interest in biology.

September 17th  National Science Popularization Day in Changsha

We cooperated with HNU-CHINA and NUDT-CHINA to participate in the Science and Innovation Fair, which helped the public to understand synthetic biology and to pay more attention to the progress and frontiers of science and technology.

September 28th  High School Presentation

We went to the AP Department of Changxian High School to inspire high school students' love for synthetic biology and enhance their understanding of cutting-edge technology.

September 30th  SynBio & SDG Science Fair at Guangzhou Science Center

Together with 11 other teams, we created a science exhibition in the world's largest science and technology center, aiming to cultivate the public's scientific literacy and educate them about synthetic biology and rare diseases.

Improved the details of our wiki.

{% endblock %}

{% extends "layout.html" %}
{% block title %}ENGINEERING SUCCESS{% endblock %}
{% block image %}

![](https://static.igem.wiki/teams/4585/wiki/engineering-success-00.png)

{% endblock %}
{% block content %}

[![](https://static.igem.wiki/teams/4585/wiki/click.png)

## System for detecting GnRH](#detecting1)

[![](https://static.igem.wiki/teams/4585/wiki/click.png)

## System for secreteing GnRH](#secreteing2)

[![](https://static.igem.wiki/teams/4585/wiki/click.png)

## Luciferase detection system](#Luciferase3)

![](https://static.igem.wiki/teams/4585/wiki/duanle.png)
![](https://static.igem.wiki/teams/4585/wiki/ccess-2.png)
![](https://static.igem.wiki/teams/4585/wiki/ccess-3.png)
![](https://static.igem.wiki/teams/4585/wiki/ccess-4.png)
![](https://static.igem.wiki/teams/4585/wiki/ccess-5.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}EXPERIMENTS{% endblock %}
{% block image %}

![](https://static.igem.wiki/teams/4585/wiki/experiments-00.png)

{% endblock %}
{% block content %}

[Plasmid mini extraction](#1Plasmid)

[Plasmid midi extraction](#2Plasmid)

[Cell rhythm detection](#3cell)

[Reverse transcription](#4Reverse)

[PCR](#5PCR)

[Homologous recombination](#6Homologous)

[Enzyme cutting and connecting](#7Enzyme)

[Transfection
(NeofectTM DNA transfection reagent)](#8Transfection)

[Transfection
 (Reagent/LipofectamineTM 3000)](#9Transfection)

[Extraction of cellular RNA](#10Extraction)

[Experiments on verification](#11Experiments)

[Western blot](#12Western)

# Plasmid mini extraction

#### 1. Heat shock transformation of Escherichia coli

![](https://static.igem.wiki/teams/4585/wiki/exx1.png)

#### 2. Mini shaking of the plasmid

![](https://static.igem.wiki/teams/4585/wiki/exx2.png)

#### 3. Plasmid mini extraction

![](https://static.igem.wiki/teams/4585/wiki/exx3.png)
![](https://static.igem.wiki/teams/4585/wiki/exx4.png)

# Plasmid midi extraction

#### 1. Heat shock transformation of Escherichia coli

![](https://static.igem.wiki/teams/4585/wiki/exx5.png)

#### 2. Midi shaking of the plasmid

![](https://static.igem.wiki/teams/4585/wiki/exx6.png)

#### 3. Plasmid midi extraction

![](https://static.igem.wiki/teams/4585/wiki/exx7.png)
![](https://static.igem.wiki/teams/4585/wiki/exx8.png)

# Cell rhythm detection

![](https://static.igem.wiki/teams/4585/wiki/exx9.png)

# Reverse transcription

#### 1. Degenerate RNA template.

![](https://static.igem.wiki/teams/4585/wiki/exx10.png)

#### 2. Configure the 1st strand cDNA synthesis reaction solution.

![](https://static.igem.wiki/teams/4585/wiki/exx11.png)

#### 3. Synthesize the 1st strand cDNA according to the following conditions.

![](https://static.igem.wiki/teams/4585/wiki/exx12.png)

# PCR

#### 1. PCR system

![](https://static.igem.wiki/teams/4585/wiki/exx13.png)
![](https://static.igem.wiki/teams/4585/wiki/exx14.png)

#### 2. PCR process

![](https://static.igem.wiki/teams/4585/wiki/exx15.png)

#### 3. Recovery scheme of PCR reaction solution

![](https://static.igem.wiki/teams/4585/wiki/exx16.png)
![](https://static.igem.wiki/teams/4585/wiki/exx17.png)

# Homologous recombination (pEASY®-Basic Seamless Cloning and AssemblyKit)

#### 1. Preparation of vectors and target fragments

![](https://static.igem.wiki/teams/4585/wiki/exx18.png)

#### 2. Establishment of cloning reaction system

![](https://static.igem.wiki/teams/4585/wiki/exx19.png)

#### 3. Transform

![](https://static.igem.wiki/teams/4585/wiki/exx20.png)

#### 4. Positive clone detection

Restriction enzymes cutting analysis of positive clones

Sequencing

# Enzyme cutting and connecting

#### 1. Enzyme cutting system

![](https://static.igem.wiki/teams/4585/wiki/exx32.png)

#### 2. Connecting

![](https://static.igem.wiki/teams/4585/wiki/exx21.png)

# Transfection (NeofectTM DNA transfection reagent)

#### 1. Cell paving board

Cells were planted in six-well plates one day in advance with a cell density of 60% -80% for transfection.

#### 2. The transfection process

![](https://static.igem.wiki/teams/4585/wiki/exx22.png)
![](https://static.igem.wiki/teams/4585/wiki/exx23.png)

# Transfection (Reagent/LipofectamineTM 3000)

![](https://static.igem.wiki/teams/4585/wiki/exx24.png)

# Extraction of cellular RNA

![Extraction of cellular RNA](https://static.igem.wiki/teams/4585/wiki/exx25.png)
![Extraction of cellular RNA](https://static.igem.wiki/teams/4585/wiki/exx26.png)

### Experiments on verification of signaling pathways through calcium flow signal changes

#### 1. Coelenterazine (Coelenterazine-h) pretreat the cells

![Experiments on verification](https://static.igem.wiki/teams/4585/wiki/exx27.png)

#### 2. Collect the cells

![Experiments on verification](https://static.igem.wiki/teams/4585/wiki/exx28.png)

#### 3. Configure the solution required

![Experiments on verification](https://static.igem.wiki/teams/4585/wiki/exx29.png)

#### 4. Equilibrate the cell suspension

![Experiments on verification](https://static.igem.wiki/teams/4585/wiki/exx30.png)

#### 5. Sirius Single-tube-type chemiluminescence detector test the Changes in the Ca2+ in cells.

(1) GnRH recombinant protein was diluted in 10 concentration gradients in a 1:3 gradient in serum-free medium, with the highest concentration of 10 µM (take 15 µL).
GnRH (10 mM) storage solution configuration: 1.1 mg GnRH powder + 104 mL ddH2O, divided into 15 µL per tube, preserve in -80℃.

(2) Turn on the power supply of the Sirius single-tube chemiluminescence detector and set it up:

(3) Set up the FB12 Sirius software.

(4) Cell activity detection:

(5) Determine the change of Ca2+ concentration in cells.

(6) After the test, save the data and clean the two pipes according to the instrument operation guide.

# Western blot

1. Prepare the cell lysate firstly (2%SDS protein lysate : Buffer/ protease inhibitor / phosphatase inhibitor = 50:1).

2. Suck off the old culture in the cell petri dish or orifice plate, add the 2%SDS cell lysate of 100ul according to the cell density and quantity. Rest on the ice for a while.After the cells fully cleaved, scratch them with the head of the gun, and finally transfer the lytic cells to the named EP tubes. And then denature the protein. According to the properties of the protein, heat the protein on a dry calorimeter at 95 ℃ for 10 minutes. After denaturation, the protein is ultrasonic on the cell ultrasonic breaker.

3. The denatured ultrasonic protein was quantified by BCA protein detection kit. Different concentrations of protein standard and protein samples to be glued were added to the enzyme plate, and BCA was added to develop color.

4. After the quantification, determine the concentration of the protein according to the absorbance, and pack the protein separately. Add the same mass protein sample to the Buffer of 2%SDS protein lysis of the same volume, and add the buffer at 10:1. Make transient centrifugation and store them in the refrigerator at -80 ℃.

5. After preparing SDS polyacrylamide gel according to the instructions, install it into the electrophoresis tank, pour into the electrophoresis solution, add the same volume of protein sample to each pore, and add protein maker. Carry out electrophoresis for 40min under 80V constant voltage. After reaching the separation gel, increase the voltage to 120V constant voltage to continue electrophoresis for 1.5h. The running position of the band is determined according to the protein maker, and the electrophoresis was stopped at the right time.

6. After labeling the PVDF membrane, activate the PVDF membrane with methanol for a few minutes, and then immerse it in the transfer solution. According to the order of blackboard + filter cotton + filter paper + glue + PVDF membrane + filter paper + filter cotton + whiteboard, install the film under the condition of constant current 290mA and maximum voltage, about 90min.

7. After the end of the film transfer, remove the film and wash off the residual transfer solution with PBST. Then seal it with 5% milk (prepared with PBST) at room temperature for 1 hour.

8. After the closure, wash the remaining milk on the film with PBST, add an antibody (prepared with 5%BSA) and immerse it. Incubate it overnight at 4 ℃.

9. The next day, recycle the used first antibody. Wash off the residual first antibody with PBST every 10min and repeat it three times. Add the second antibody of the same species (prepared with 5% milk) and incubate at room temperature for 1 hour. Wash off the residual secondary antibody with PBST every 10min and replace it three times.

10. Develop on the strip developer and save the picture. The strips can be stored in the refrigerator at-20 ℃.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block image %}

![](https://static.igem.wiki/teams/4585/wiki/results-00.png)

{% endblock %}
{% block content %}

![](https://static.igem.wiki/teams/4585/wiki/zasi-1.png)
![](https://static.igem.wiki/teams/4585/wiki/zasi-2.png)
![](https://static.igem.wiki/teams/4585/wiki/zasi-3.png)
![](https://static.igem.wiki/teams/4585/wiki/zasi-4.png)
![](https://static.igem.wiki/teams/4585/wiki/zasi-5.png)

{% endblock %}