iGEM 2018 — Team ECUST

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# Team:ECUST/Notebook

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* [HOME](https://2018.igem.org/Team%3AECUST)
* [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [ATTRIBUTION](https://2018.igem.org/Team%3AECUST/Attributions)
  + [SPONSORS](https://2018.igem.org/Team%3AECUST/Sponsors)
* [PROJECT](https://2018.igem.org/Team%3AECUST/Description)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Description)
  + [BACKGROUND](https://2018.igem.org/Team%3AECUST/Background)
  + [DESIGN](https://2018.igem.org/Team%3AECUST/Design)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Experiments)
  + [RUST REMOVER](https://2018.igem.org/Team%3AECUST/Rust_Remover)
  + [BIOFILM REMOVER](https://2018.igem.org/Team%3AECUST/Biofilm_Remover)
  + [FUR INVERTER](https://2018.igem.org/Team%3AECUST/Fur_Inverter)
  + [LYSIN](https://2018.igem.org/Team%3AECUST/Lysin)
  + [BIOCIDE](https://2018.igem.org/Team%3AECUST/Biocide)
  + [LIGHT-ON SUICIDE](https://2018.igem.org/Team%3AECUST/Light-on_Suicide)
  + [DEMONSTRATION](https://2018.igem.org/Team%3AECUST/Demonstrate)
* [PARTS](https://2018.igem.org/Team%3AECUST/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3AECUST/Parts#section1)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3AECUST/Parts#section2)
* [MODEL](https://2018.igem.org/Team%3AECUST/Model)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Model)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Quorum_Sensing)
  + [IRON SENSING](https://2018.igem.org/Team%3AECUST/Iron_Sensing)
* [DEVICE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [HARDWARE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [SOFTWARE](https://2018.igem.org/Team%3AECUST/Software)
* [HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [INTEGRATED HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Integrated)
  + [COMMUNICATION](https://2018.igem.org/Team%3AECUST/Communication)
  + [EDUCATION & PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AECUST/Public_Engagement)
  + [APPLIED DESIGN](https://2018.igem.org/Team%3AECUST/Applied_Design)
  + [COLLABORATION](https://2018.igem.org/Team%3AECUST/Collaborations)
* [BIOSAFETY](https://2018.igem.org/Team%3AECUST/Safety)
* [NOTEBOOK](https://2018.igem.org/Team%3AECUST/Notebook)
  + [PROTOCOL](https://2018.igem.org/Team%3AECUST/Protocol)
* [JUDGING FORM](https://igem.org/2018_Judging_Form?id=2737)
* [INTERLAB](https://2018.igem.org/Team%3AECUST/InterLab)

Notebook

# April

## Week1 (4.1-4.7)

We purchased the *iron bacteria* from the Chinese Academy of Sciences strain library.Although we tried to culture the *iron bacteria*, we failed.

## Week2(4.8-4.14)

We decided to screen the *iron bacteria* from the nature,so we put the iron into the lack and hope that rust can form in a short time.

## Week3(4.15-4.21)

That’s a good news.The *iron bacteria* were screened from rusty iron sample and cultured by Winogradsky culture medium.

## Week4(4.22-4.28)

We start to construct quorum sensing part of the gene circuit. The DNA fragment,AfeR-GFP was synthesized by Genscript, and GFP was chosen as our reporter gene.

## Week5(4.29-5.5)

We were still busy with the construction of the recombinant plasmid. Because the difficulty of the work that we digested DNA fragment by restriction endonuclease and linked them with T4 ligase/SolutionⅠwas really beyond our imagination.

# May

## Week6(5.6-5.12)

This we tried to use Gibson assembly to overcome the last week failure.And we did it!We have to say Gibson assembly is a real convenient and efficient way.

## Week7(5.13-5.19)

We characterized our interested fragment by detecting the fluorescence value of GFP under the different concentration of AHL, a signal molecule.

## Week8(5.20-5.26)

Our gene loop consists of many parts, and this week we constructed the expression vector of Dispersin B, whose promoter is inducted by the complex AfeR-AHL.

## Week9(5.27-6.2)

As mentioned above, Gibson assembly is more easy than the traditional method in construction work.However,we can’t use Gibson assembly in this part.That’s because there is Dispersin B’s coding gene in the genome of *E.coli*.So, you will see we would do the same thing in the following weeks until we succeed

# June

## Week10(6.3-6.9)

Construction pET-28a(+)-dspB

## Week11(6.10-6.16)

We picked out some positive clones.Adding IPTG, culture overnight at 25℃.Then we broke these bacterial and obtained supernatant to do antibiofilm experiments and activity measurement.Also, the construction of enterobactin, a kind fo siderophore,was going on.

## Week12(6.17-6.23)

It was the hardest part we have ever constructed.Because the cloned fragment was so long that we have to carry out several rounds of PCR.

## Week13(6.24-6.30)

Our work was not going anywhere, so we asked for help from our instructor. With some discussion and attempt, we find some ways to solve problems.

# July

## Week14(7.1-7.7)

Continue the work.

## Week15(7.8-7.14)

This week, we succeeded in inserting a gene cluster of enterobactin into vector pET-28a(+). After that, we get this supernatant to measure its ability of removing ferric iron.

## Week16(7.15-7.21)

We borrowed Light-on plasmid and its host bacteria,3J1 from Yang’s laboratory, and we thank Prof Yang Yi from East China University of Science and Technology for giving us the plasmid. We cultured 3J1 and measured growth curve.

## Week17(7.22-7.28)

We constructed Light-on-mCherry, and measured fluorescence intensity under different conditions.(7.29-8.4)

# August

## Week18(8.5-8.11)

Due to the long period of expression of mCherry and some invalid data, we spent this week on doing the same thing as last week.Apart from this, we built a model by these data

## Week19(8.12-8.18)

Base on the last two weeks’ work, we construct Light-on-Mnase.

## Week20(8.19-8.25)

We divided these bacteria into two groups.One is cultured in the dark. The other is under the light.We measured OD600 to determine cell viability.

## Week21(8.26-9.1)

Build ,model about light suicide

# September

## Week22(9.2-9.8)

We designed and synthesized three kinds of promoters, fur1,fur2 and fur3 which were inserted fur box.It cost some time to get these samples.

## Week23(9.9-9.15)

We characterize these modified promoters by measuring the fluorescence intensity of mCherry.

## Week24(9.16-9.22)

Based on the last week, we find that fur2 has the lowest leakage expression of lacI under low Fe concentration and the highest expression under high Fe concentration.Therefore we chosen fur2 promoter to use in our system.Then we constructed fur2-AD to realise that E.coli can express ceropin AD under high iron ion concentration.

## Week25(9.23-9.29)

We observed and evaluated the expression product, when synthesized AD is used as a positive control.

# October

## Week26(9.30-10.6)

We construct fur2-lysin to achieve that E.coli can express lysin under IPTG induction.

## Week27(10.6-10.13)

We finished the characterization of lysin by measuring the light absorption of 600nm.Meanwhile, we also use normal temperature scanning electron microscope to observe the effect of lysin. In addition to this,the Effects of killing *iron bacteria* with antibacterial peptide, DspB and siderophore were considered comprehensively.

Bring me up!

[![](https://static.igem.org/mediawiki/2018/8/8c/T--ECUST--footer1.png "EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY")](https://www.ecust.edu.cn)
[![](https://static.igem.org/mediawiki/2018/b/b8/T--ECUST--footer2.png "School of Biotechnology")](http://biotech.ecust.edu.cn)

| [![](https://static.igem.org/mediawiki/2018/2/2c/T--ECUST--footer4.png)](https://www.google.com/maps/place/East%2BChina%2BUniversity%2Bof%2BScience%2Band%2BTechnology%2B%EF%BC%88South%2BGate%EF%BC%89/%4031.1409016%2C121.4230593%2C17z/data%3D%213m1%214b1%214m5%213m4%211s0x35b2635fa3d40407%3A0xa2e03588e259541f%218m2%213d31.140897%214d121.425248) | iGEM-Team ECUST EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY  Shanghai China |

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# Team:ECUST/Experiments

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* [HOME](https://2018.igem.org/Team%3AECUST)
* [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [ATTRIBUTION](https://2018.igem.org/Team%3AECUST/Attributions)
  + [SPONSORS](https://2018.igem.org/Team%3AECUST/Sponsors)
* [PROJECT](https://2018.igem.org/Team%3AECUST/Description)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Description)
  + [BACKGROUND](https://2018.igem.org/Team%3AECUST/Background)
  + [DESIGN](https://2018.igem.org/Team%3AECUST/Design)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Experiments)
  + [RUST REMOVER](https://2018.igem.org/Team%3AECUST/Rust_Remover)
  + [BIOFILM REMOVER](https://2018.igem.org/Team%3AECUST/Biofilm_Remover)
  + [FUR INVERTER](https://2018.igem.org/Team%3AECUST/Fur_Inverter)
  + [LYSIN](https://2018.igem.org/Team%3AECUST/Lysin)
  + [BIOCIDE](https://2018.igem.org/Team%3AECUST/Biocide)
  + [LIGHT-ON SUICIDE](https://2018.igem.org/Team%3AECUST/Light-on_Suicide)
  + [DEMONSTRATION](https://2018.igem.org/Team%3AECUST/Demonstrate)
* [PARTS](https://2018.igem.org/Team%3AECUST/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3AECUST/Parts#section1)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3AECUST/Parts#section2)
* [MODEL](https://2018.igem.org/Team%3AECUST/Model)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Model)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Quorum_Sensing)
  + [IRON SENSING](https://2018.igem.org/Team%3AECUST/Iron_Sensing)
* [DEVICE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [HARDWARE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [SOFTWARE](https://2018.igem.org/Team%3AECUST/Software)
* [HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [INTEGRATED HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Integrated)
  + [COMMUNICATION](https://2018.igem.org/Team%3AECUST/Communication)
  + [EDUCATION & PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AECUST/Public_Engagement)
  + [APPLIED DESIGN](https://2018.igem.org/Team%3AECUST/Applied_Design)
  + [COLLABORATION](https://2018.igem.org/Team%3AECUST/Collaborations)
* [BIOSAFETY](https://2018.igem.org/Team%3AECUST/Safety)
* [NOTEBOOK](https://2018.igem.org/Team%3AECUST/Notebook)
  + [PROTOCOL](https://2018.igem.org/Team%3AECUST/Protocol)
* [JUDGING FORM](https://igem.org/2018_Judging_Form?id=2737)
* [INTERLAB](https://2018.igem.org/Team%3AECUST/InterLab)

Quorum Sensing

# Description

Quorum sensing is an organic chemical signal that regulates a variety of physiological activities of bacteria, such as cell movement, growth, and biofilm formation. This sensing mechanism is mediated by diffusible chemical signals called homoserine lactones. The concentration of signal molecules outside the cell increases with increasing cell density and the signal molecule itself is permeable to the cell membrane. Upon reaching the threshold concentration of the chemical signal, the quorum sensing system is activated to trigger expression or inhibition of the gene.

![](https://static.igem.org/mediawiki/2018/3/32/T--ECUST--quo_sen1.png)

# Design

We used the quorum sensing system of *iron bacteria* isolated from pipeline rust. This system is homologous to the LuxR system. The *iron bacteria* itself produces the AfeR protein, which binds to 3-oxo-C12-AHL and dimerizes. This dimer binds to afeR box and activates gene transcription. Therefore, the recognition of *iron bacteria* by *Escherichia coli* can be achieved by constructing a heterologous AfeR protein of *iron bacteria* and a corresponding activated promoter in engineered *E. coli*.

![Figure1.1 OD600 reference point tab](https://static.igem.org/mediawiki/2018/8/8a/T--ECUST--quo_sen2.png)

![Figure1.1 OD600 reference point tab](https://static.igem.org/mediawiki/2018/3/3c/T--ECUST--quo_sen3.png)

# Result

Gene fragment of GFP is synthesized through Genescript and it is on vector pUC57.

![](https://static.igem.org/mediawiki/2018/e/e4/T--ECUST--QUORUM_SENSING_F1.jpeg)

**Figure 1. Insert GFP(controlled by promoter with Afe-box) to vector pUC57.**

The plasmid was transformed to *E. coli* DH5α and cultured at 37 °C for 12 h. Then positive bacteria is cultured in LB culture medium adding with 0.1% ampicillin. After bacteria grows to logarithmic phase, adding AHL until final concentration is 10-4 ,10-5,10-6 ,10-7,10-8, 10-9 mol/L. Then light absorption is measured. Excitation/Emission wavelength is 485nm/530nm.

![](https://static.igem.org/mediawiki/2018/c/cb/T--ECUST--QUORUM_SENSING_F2.jpg)

**Figure 2. (a) Fluorescence intensity at different time induced by different concentrations of AHL.
(b)A three-dimensional map with log10 concentration of AHL,time and fluorescence intensity as the coordinate**

When the AHL concentration is in the range of 10-7~10-8 mol/L, the fluorescence insensity was jump. When the AHL concentration is above 10-7 mol/L, the promoter will respond.

# Reference

1. Mol Syst Biol. 2011; 7: 521. Engineering microbes to sense and eradicate *Pseudomonas aeruginosa*, a human pathogen Nazanin Saeidi,1,\* Choon Kit Wong

2. Biological Research 2005 A Lux-like quorum sensing system in the extreme acidophile *Acidithiobacillus ferrooxidans*. Mariella O. Rivas, Michael Seeger

3.American Society for Microbiology Evidence for a Functional Quorum-Sensing Type AI-1 System in the Extremophilic Bacterium *Acidithiobacillus ferrooxidans* Carolina Farah1, Mario Vera

Bring me up!

[![](https://static.igem.org/mediawiki/2018/8/8c/T--ECUST--footer1.png "EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY")](https://www.ecust.edu.cn)
[![](https://static.igem.org/mediawiki/2018/b/b8/T--ECUST--footer2.png "School of Biotechnology")](http://biotech.ecust.edu.cn)

| [![](https://static.igem.org/mediawiki/2018/2/2c/T--ECUST--footer4.png)](https://www.google.com/maps/place/East%2BChina%2BUniversity%2Bof%2BScience%2Band%2BTechnology%2B%EF%BC%88South%2BGate%EF%BC%89/%4031.1409016%2C121.4230593%2C17z/data%3D%213m1%214b1%214m5%213m4%211s0x35b2635fa3d40407%3A0xa2e03588e259541f%218m2%213d31.140897%214d121.425248) | iGEM-Team ECUST EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY  Shanghai China |

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# Team:ECUST/Results

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* [HOME](https://2018.igem.org/Team%3AECUST)
* [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [TEAM](https://2018.igem.org/Team%3AECUST/Team)
  + [ATTRIBUTION](https://2018.igem.org/Team%3AECUST/Attributions)
  + [SPONSORS](https://2018.igem.org/Team%3AECUST/Sponsors)
* [PROJECT](https://2018.igem.org/Team%3AECUST/Description)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Description)
  + [BACKGROUND](https://2018.igem.org/Team%3AECUST/Background)
  + [DESIGN](https://2018.igem.org/Team%3AECUST/Design)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Experiments)
  + [RUST REMOVER](https://2018.igem.org/Team%3AECUST/Rust_Remover)
  + [BIOFILM REMOVER](https://2018.igem.org/Team%3AECUST/Biofilm_Remover)
  + [FUR INVERTER](https://2018.igem.org/Team%3AECUST/Fur_Inverter)
  + [LYSIN](https://2018.igem.org/Team%3AECUST/Lysin)
  + [BIOCIDE](https://2018.igem.org/Team%3AECUST/Biocide)
  + [LIGHT-ON SUICIDE](https://2018.igem.org/Team%3AECUST/Light-on_Suicide)
  + [DEMONSTRATION](https://2018.igem.org/Team%3AECUST/Demonstrate)
* [PARTS](https://2018.igem.org/Team%3AECUST/Parts)
  + [BASIC PARTS](https://2018.igem.org/Team%3AECUST/Parts#section1)
  + [COMPOSITE PARTS](https://2018.igem.org/Team%3AECUST/Parts#section2)
* [MODEL](https://2018.igem.org/Team%3AECUST/Model)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Model)
  + [QUORUM SENSING](https://2018.igem.org/Team%3AECUST/Quorum_Sensing)
  + [IRON SENSING](https://2018.igem.org/Team%3AECUST/Iron_Sensing)
* [DEVICE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [HARDWARE](https://2018.igem.org/Team%3AECUST/Hardware)
  + [SOFTWARE](https://2018.igem.org/Team%3AECUST/Software)
* [HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [OVERVIEW](https://2018.igem.org/Team%3AECUST/Human_Practices)
  + [INTEGRATED HUMAN PRACTICE](https://2018.igem.org/Team%3AECUST/Integrated)
  + [COMMUNICATION](https://2018.igem.org/Team%3AECUST/Communication)
  + [EDUCATION & PUBLIC ENGAGEMENT](https://2018.igem.org/Team%3AECUST/Public_Engagement)
  + [APPLIED DESIGN](https://2018.igem.org/Team%3AECUST/Applied_Design)
  + [COLLABORATION](https://2018.igem.org/Team%3AECUST/Collaborations)
* [BIOSAFETY](https://2018.igem.org/Team%3AECUST/Safety)
* [NOTEBOOK](https://2018.igem.org/Team%3AECUST/Notebook)
  + [PROTOCOL](https://2018.igem.org/Team%3AECUST/Protocol)
* [JUDGING FORM](https://igem.org/2018_Judging_Form?id=2737)
* [INTERLAB](https://2018.igem.org/Team%3AECUST/InterLab)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

[![](https://static.igem.org/mediawiki/2018/8/8c/T--ECUST--footer1.png "EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY")](https://www.ecust.edu.cn)
[![](https://static.igem.org/mediawiki/2018/b/b8/T--ECUST--footer2.png "School of Biotechnology")](http://biotech.ecust.edu.cn)

| [![](https://static.igem.org/mediawiki/2018/2/2c/T--ECUST--footer4.png)](https://www.google.com/maps/place/East%2BChina%2BUniversity%2Bof%2BScience%2Band%2BTechnology%2B%EF%BC%88South%2BGate%EF%BC%89/%4031.1409016%2C121.4230593%2C17z/data%3D%213m1%214b1%214m5%213m4%211s0x35b2635fa3d40407%3A0xa2e03588e259541f%218m2%213d31.140897%214d121.425248) | iGEM-Team ECUST EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY  Shanghai China |

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