iGEM 2019 — Team iBowu-China

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# Team:iBowu-China/Notebook

iBowu-China

* [iBowu-China](index.html)
* [Judging Form](https://igem.org/2019_Judging_Form?team=iBowu-China)
* [Team](https://2019.igem.org/Team%3AiBowu-China/Team)
* [Safety](https://2019.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2019.igem.org/Team%3AiBowu-China/Human_Practices)
* [Modeling](https://2019.igem.org/Team%3AiBowu-China/Modeling)
* [Project Design](https://2019.igem.org/Team%3AiBowu-China/Project_Design)

  [InterLab Study](https://2019.igem.org/Team%3AiBowu-China/InterLab_Study)
  [Notebook](https://2019.igem.org/Team%3AiBowu-China/Notebook)
  [Protocals](https://2019.igem.org/Team%3AiBowu-China/Protocals)
  [Result](https://2019.igem.org/Team%3AiBowu-China/Result)

- [Judging Form](https://igem.org/2019_Judging_Form?team=iBowu-China)
- [Team](team.html)
- [Human Practices](hp.html)
- [Modeling](modeling.html)
- [Project Design](pd.html)

  [InterLab](interlab.html)
  [Notebook](notebook.html)
  [Protocals](protocals.html)
  [Result](result.html)

Phactory

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# Team Project Abstract

Potatoes are the fourth largest crops in the world. Soft rot is one of the main plant diseases for the decrease
of potato production. Pectobacterium carotovorum (Erwinia carotovorum) is a main plant pathogen causing soft rot
of potatoes as well as many other crops. We plan to develop an easily-used toolkit based on cell-free system
which can detect and prevent the occurrence of soft rot. Two kinds of gene circuits are designed for the
detection and prevention. One is to detect AHL (quorum sensing signal molecular) of P. carotovorum; the other is
to express the hydrolase AiiA and the antibacterial peptides which could degrade AHL and generally kill bacteria
separately. The gene circuits coupling with cell-free expression system are lyophilized on paper and can be used
with rehydration.

# Project Design

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Project Design Details](project.html)

# Human Practices

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Human Practices Details](hp.html)

# Modeling

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Modeling Details](modeling.html)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:iBowu-China/Experiments

iBowu-China

* [iBowu-China](index.html)
* [Judging Form](https://igem.org/2019_Judging_Form?team=iBowu-China)
* [Team](https://2019.igem.org/Team%3AiBowu-China/Team)
* [Safety](https://2019.igem.org/Team%3AiBowu-China/Safety)
* [Human Practices](https://2019.igem.org/Team%3AiBowu-China/Human_Practices)
* [Modeling](https://2019.igem.org/Team%3AiBowu-China/Modeling)
* [Project Design](https://2019.igem.org/Team%3AiBowu-China/Project_Design)

  [InterLab Study](https://2019.igem.org/Team%3AiBowu-China/InterLab_Study)
  [Notebook](https://2019.igem.org/Team%3AiBowu-China/Notebook)
  [Protocals](https://2019.igem.org/Team%3AiBowu-China/Protocals)
  [Result](https://2019.igem.org/Team%3AiBowu-China/Result)

- [Judging Form](https://igem.org/2019_Judging_Form?team=iBowu-China)
- [Team](team.html)
- [Human Practices](hp.html)
- [Modeling](modeling.html)
- [Project Design](pd.html)

  [InterLab](interlab.html)
  [Notebook](notebook.html)
  [Protocals](protocals.html)
  [Result](result.html)

Phactory

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[Previous](#myCarousel)
[Next](#myCarousel)

# Team Project Abstract

Potatoes are the fourth largest crops in the world. Soft rot is one of the main plant diseases for the decrease
of potato production. Pectobacterium carotovorum (Erwinia carotovorum) is a main plant pathogen causing soft rot
of potatoes as well as many other crops. We plan to develop an easily-used toolkit based on cell-free system
which can detect and prevent the occurrence of soft rot. Two kinds of gene circuits are designed for the
detection and prevention. One is to detect AHL (quorum sensing signal molecular) of P. carotovorum; the other is
to express the hydrolase AiiA and the antibacterial peptides which could degrade AHL and generally kill bacteria
separately. The gene circuits coupling with cell-free expression system are lyophilized on paper and can be used
with rehydration.

# Project Design

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Project Design Details](project.html)

# Human Practices

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Human Practices Details](hp.html)

# Modeling

--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

 [Modeling Details](modeling.html)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:iBowu-China/Results

Sticky Animated Navigation Bar

[![](https://static.igem.org/mediawiki/2019/f/f2/T--iBowu-China--logo6666.jpg)](https://2019.igem.org/Team%3AiBowu-China)

Project

[Description](https://2019.igem.org/Team%3AiBowu-China/Description)
[Design](https://2019.igem.org/Team%3AiBowu-China/Design)
[Results](https://2019.igem.org/Team%3AiBowu-China/Demonstrate)

[Parts](https://2019.igem.org/Team%3AiBowu-China/Parts)

Interlab

[Notebook](https://2019.igem.org/Team%3AiBowu-China/NoteBook)
[Protocols](https://2019.igem.org/Team%3AiBowu-China/Protocols)
[Safety](https://2019.igem.org/Team%3AiBowu-China/Safety)

Human Practices

[Integrated HP](https://2019.igem.org/Team%3AiBowu-China/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AiBowu-China/Public_Engagement)
[Entrepreneurship](https://2019.igem.org/Team%3AiBowu-China/Entrepreneurship)

Modeling

[Model](https://2019.igem.org/Team%3AiBowu-China/Model)
[Software](https://2019.igem.org/Team%3AiBowu-China/Software)

[Judging form](https://2019.igem.org/Team%3AiBowu-China/Judging)

Team

[Team members](https://2019.igem.org/Team%3AiBowu-China/team)
[Collaborations](https://2019.igem.org/Team%3AiBowu-China/Collaborations)
[Attributions](https://2019.igem.org/Team%3AiBowu-China/Attributions)

![](https://static.igem.org/mediawiki/2019/2/22/T--iBowu-China--bjmeetuppic1.jpeg)

[Table of Contents](#TOC)
:   [Result Demonstration](#ResultDemo)
:   [Construction of Quorum Snesing Genetic Circuits](#ResultConstruction)
:   [Selection of a Better Sensing Circuit with Different AHL Receptors](#ResultSelection)
:   [Development of Cell-free Systems](#ResultCellFree)
:   [Establishment and Application to the Soft Rot Bio-sense and Bio-control](#ResultEstablishment)
:   [Verification of the Bio-control System to Soft Rot](#ResultVerify)

Result Demonstration

* Selecting a sensitive E. carotovora AHL sensing system
* Developing cell-free a quorum sensing signal AHL detection platform
* Establishing a testing strip for the detection of AHL

In our project, we characterized four quorum sensing system for the pathogenic detection and applied to a novel testing strip that could detect the EC quorum sensing signals effectively. Therefore, we performed experiments on five parts and the experimental characterizations of our detection systems are shown as follows.

Construction of Quorum Sensing Genetic Circuits

We selected four bacterial quorum sensing systems for the E. carotovora pathogenic detecting and controlling. Three quorum sensing signal (OHHL) receptors of E. carotovora and the common known receptor LuxR were chosen for the soft rot sensing elements. ExpR153, Exp71, and EcbR are the AHL receptors of three E. carotovora subsp. respectively. And LuxR was the AHL receptor of Vibrio fischeri. The sensing gene circuits were constructed as the following design (Fig. 1):

1. J23101- ExpR153 -pExp-GFP
2. J23101- Exp71-pExp-GFP
3. J23101- EcbR - pExp-GFP
4. J23101- LuxR- pLux-GFP
5. J23101- ExpR153 -pExp- LacZ
6. J23101- Exp71-pExp- LacZ
7. J23101- EcbR - pExp- LacZ
8. J23101- LuxR- pLux-LacZ

The four receptors (ExpR153, Exp71, EcbR, and LuxR) were constitutive expressed under a constitutive promoter down streamed with the pExp/pLux regulating reporters (GFP and LacZ).

The construction of circuits was competed by Gibson Assembly, and confirmed by sequencing.

![](https://static.igem.org/mediawiki/2019/3/3b/T--iBowu-China--Results1.png)
![](https://static.igem.org/mediawiki/2019/5/52/T--iBowu-China--Results2.png)

Figure 1. Schematic diagram of gene circuits. The four receptors (ExpR153, Exp71, EcbR, and LuxR) were constitutive expressed under a constitutive promoter down streamed with the pExp/pLux regulating reporters (GFP and LacZ).

Selection of a Better Sensing Circuit with Different AHL Receptors

After the construction of the circuits, we first characterized the cellular sensing features within E.coli. The circuits were transformed into E.coli. It can be visible to the color of green because of the high expression of GFP after incubation for a period of time. That might be attributed to the quorum sensing of E.coli. The engineered bacteria were incubated in the plate reader and measured the OD600 for cell density and fluorescent intensity every 5 min for two hours, and the inducer AHL was added after 1 h.

Before the induction, it showed that there was no expression of the engineered bacteria with gene circuits of pcon-Exp71-pExp-GFP and pcon-LuxR-pLux-GFP. The intensity of bacteria was not intensive enough for the inducing expression. For the bacteria with pcon-Exp71-pExp-GFP, it seemed that there was fluorescence output after 20 min incubation. the bacteria intensity would course quorum sensing in this circuit. For pcon-Exp153-pExp-GFP, the fluorescent protein was expressed from the start, which showed a sensitive binding of inducer.

![](https://static.igem.org/mediawiki/2019/0/01/T--iBowu-China--ResultsGraph1.png)

Figure 2. The fluorescence of engineering bacteria with four gene circuits in the 100 min incubation. The inducer AHL was added to the culture after 60 min incubation.
After the AHL induction, there expressed the fluorescent protein within the circuit of LuxR. And the 1 nM AHL could course the regulated expression. However, the circuit of Exp71 still showed no expression (Fig. 3).

![](https://static.igem.org/mediawiki/2019/0/04/T--iBowu-China--ResultsGraph2.png)
![](https://static.igem.org/mediawiki/2019/3/33/T--iBowu-China--ResultsGraph3.png)

Figure 3. The fluorescence of three engineered bacteria expressed after AHL induction.

Development of Cell-free Systems

After the cellular sensing characterization, we applied our gene circuits to the cell-free systems. The four gene circuits were characterized in cell-free system with a series of inducer (OHHL) concentrations, respectively. Although the quorum sensing occurred in cellular expression, it seemed that there still were some challenges to transfer the detection system to cell-free expression system. After the overnight cell-free inducing incubation, it showed that there only the circuit of LuxR-sfGFP was response to AHL with the detection limit of 1 nM. The invalid detection of E. carotovora receptor-based system might be contributed to the noneffective expression in cell-free system. In this detection system, no more than 10 nM of AHL could induce the strong fluorescence output (Fig. 4).

![](https://static.igem.org/mediawiki/2019/2/28/T--iBowu-China--ResultsGraph4.png)

Figure 4. The cell-free fluorescence output induced by a series of AHL concentrations.

Establishment and Application to the Soft Rot Bio-sense and Bio-control

Based on the cell-free induction results, we placed the quorum sensing system to papers by freeze-drying technology. The reporters were replaced by the visible signal protein LacZ. LacZ is β-galactosidase which can cleave the yellow substrate to purple. The four gene circuits were tested to AHL inducing experiments.

Similar to the previous results, only the LuxR-LacZ gene circuit was successfully detecting the AHL. Among the series of AHL concentrations induction, it showed that the detection limit was lower than 1 nM. Moreover, there is a significant visible output with 10 nm AHL treatment (Fig. 5).

![](https://static.igem.org/mediawiki/2019/c/c4/T--iBowu-China--Resultsgraph5.png)

Figure 5. The detection limit and detection time of quorum sensing based testing strips.

Verification of the Bio-control System to Soft Rot

After the detecting the pathogenic bacteria E. carotovora, we tried to treat the soft rot with our biological methods.