iGEM 2020 — Team TPR_China

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# Team:TPR China/Notebook

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Integrated Human Practice - Team:TPR\_China - 2020.igem.org

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###### Home

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###### Team

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###### Project

![](https://static.igem.org/mediawiki/2020/thumb/5/56/T--TPR_China--results.png/180px-T--TPR_China--results.png)

###### Results

![](https://static.igem.org/mediawiki/2020/9/9d/T--TPR_China--Judging.png)

###### Judging

![](https://static.igem.org/mediawiki/2020/0/0d/T--TPR_China--part.png)

###### Part

![](https://static.igem.org/mediawiki/2020/thumb/0/07/T--TPR_China--HP_logo.png/180px-T--TPR_China--HP_logo.png)

###### HP

![](https://static.igem.org/mediawiki/2020/e/ed/T--TPR_China--Lab.png)

###### Lab

# Home

# Team

[TeamMembers](https://2020.igem.org/Team%3ATPR_China/TeamMembers)
[Attributions](https://2020.igem.org/Team%3ATPR_China/Attributions)
[Acknowledgement](https://2020.igem.org/Team%3ATPR_China/Acknowledgement)
[Collaborations](https://2020.igem.org/Team%3ATPR_China/Collaborations)
[Partnership](https://2020.igem.org/Team%3ATPR_China/Partnership)

# Project

[Description](https://2020.igem.org/Team%3ATPR_China/Description)
[Design](https://2020.igem.org/Team%3ATPR_China/Design)
[Implementation](https://2020.igem.org/Team%3ATPR_China/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ATPR_China/Proof_Of_Concept)
[Contribution](https://2020.igem.org/Team%3ATPR_China/Contribution)

# Results

[Experiment](https://2020.igem.org/Team%3ATPR_China/Experiment)
[Engineering](https://2020.igem.org/Team%3ATPR_China/Engineering)
[Model](https://2020.igem.org/Team%3ATPR_China/Model)

# Judging

# Part

[Improvement](https://2020.igem.org/Team%3ATPR_China/Improvement)
[Composite](https://2020.igem.org/Team%3ATPR_China/Composite)
[Collection](https://2020.igem.org/Team%3ATPR_China/Collection)
[Basic](https://2020.igem.org/Team%3ATPR_China/Basic)

# HP

[IHP](https://2020.igem.org/Team%3ATPR_China/Human_Practices)
[ScienceCommunication](https://2020.igem.org/Team%3ATPR_China/Education)
[Inclusion](https://2020.igem.org/Team%3ATPR_China/Inclusion)

# Lab

[Notebook](https://2020.igem.org/Team%3ATPR_China/Notebook)
[Protocol](https://2020.igem.org/Team%3ATPR_China/Protocol)
[Safety](https://2020.igem.org/Team%3ATPR_China/Safety)

# Home

###### Our Home

# Team

###### Our team, TPR\_China, consists of students from several high schools, several team advisers and academic mentors. Our team members come from different cities in China and United States. The diversity makes our team unique in culture and broad in knowledge base.

# Project

###### Hope to know new methods to contain the locust problem? Click this link to see the unique project of our team and to know more about the locust master.

# Results

###### Successfully constructed engineering bacteria in to produce nitrilase with enough enzyme activity.

# Judging

###### Why do we deserve metals, honors, and awards? Why are we excellent? We hope to help judges on evaluating our project.

# Part

###### We have interesting part and we want to share them with you. Parts including several Sequences of nitrilases and plasmids constructed are applied by our team, including originals and new parts improved and collected based on the originals.

# Human Practices

###### Besides working in the lab and conducing research, we also pay attention to human practices in order to raise social awareness of synthetic biology and advocate our projects and solutions.

# Lab

###### This section includes notebook, general protocols, as well as lab safety during the process of experiments.

###### Notebook

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TPR China/Engineering

<!DOCTYPE html>

Integrated Human Practice - Team:TPR\_China - 2020.igem.org

![](/wiki/images/5/5c/T--TPR_China--backtopimg3.png)

![](/wiki/images/thumb/c/c4/T--TPR_China--index_logo.png/180px-T--TPR_China--index_logo.png)

###### Home

![](/wiki/images/thumb/8/8c/T--TPR_China--teamlogo.png/180px-T--TPR_China--teamlogo.png)

###### Team

![](/wiki/images/thumb/4/41/T--TPR_China--projectlogo.png/180px-T--TPR_China--projectlogo.png)

###### Project

![](/wiki/images/thumb/5/56/T--TPR_China--results.png/180px-T--TPR_China--results.png)

###### Results

![](https://static.igem.org/mediawiki/2020/9/9d/T--TPR_China--Judging.png)

###### Judging

![](/wiki/images/0/0d/T--TPR_China--part.png)

###### Part

![](/wiki/images/thumb/0/07/T--TPR_China--HP_logo.png/180px-T--TPR_China--HP_logo.png)

###### HP

![](/wiki/images/e/ed/T--TPR_China--Lab.png)

###### Lab

# Home

# Team

[TeamMembers](https://2020.igem.org/Team%3ATPR_China/TeamMembers)
[Attributions](https://2020.igem.org/Team%3ATPR_China/Attributions)
[Acknowledgement](https://2020.igem.org/Team%3ATPR_China/Acknowledgement)
[Collaborations](https://2020.igem.org/Team%3ATPR_China/Collaborations)
[Partnership](https://2020.igem.org/Team%3ATPR_China/Partnership)

# Project

[Description](https://2020.igem.org/Team%3ATPR_China/Description)
[Design](https://2020.igem.org/Team%3ATPR_China/Design)
[Implementation](https://2020.igem.org/Team%3ATPR_China/Implementation)
[Proof of Concept](https://2020.igem.org/Team%3ATPR_China/Proof_Of_Concept)
[Contribution](https://2020.igem.org/Team%3ATPR_China/Contribution)

# Results

[Experiment](https://2020.igem.org/Team%3ATPR_China/Experiment)
[Engineering](https://2020.igem.org/Team%3ATPR_China/Engineering)
[Model](https://2020.igem.org/Team%3ATPR_China/Model)

# Judging

# Part

[Improvement](https://2020.igem.org/Team%3ATPR_China/Improvement)
[Composite](https://2020.igem.org/Team%3ATPR_China/Composite)
[Collection](https://2020.igem.org/Team%3ATPR_China/Collection)
[Basic](https://2020.igem.org/Team%3ATPR_China/Basic)

# HP

[IHP](https://2020.igem.org/Team%3ATPR_China/Human_Practices)
[ScienceCommunication](https://2020.igem.org/Team%3ATPR_China/Education)
[Inclusion](https://2020.igem.org/Team%3ATPR_China/Inclusion)

# Lab

[Notebook](https://2020.igem.org/Team%3ATPR_China/Notebook)
[Protocol](https://2020.igem.org/Team%3ATPR_China/Protocol)
[Safety](https://2020.igem.org/Team%3ATPR_China/Safety)

# Home

###### Our Home

# Team

###### Our team, TPR\_China, consists of students from several high schools, several team advisers and academic mentors. Our team members come from different cities in China and United States. The diversity makes our team unique in culture and broad in knowledge base.

# Project

###### Hope to know new methods to contain the locust problem? Click this link to see the unique project of our team and to know more about the locust master.

# Results

###### Successfully constructed engineering bacteria in to produce nitrilase with enough enzyme activity.

# Judging

###### Why do we deserve metals, honors, and awards? Why are we excellent? We hope to help judges on evaluating our project.

# Part

###### We have interesting part and we want to share them with you. Parts including several Sequences of nitrilases and plasmids constructed are applied by our team, including originals and new parts improved and collected based on the originals.

# Human Practices

######

# Lab

###### This section includes notebook, general protocols, as well as lab safety during the process of experiments.

###### Engineering

## Overview

We decided to use nitrilase - a protein that degrades nitriles - for degradation of PAN.

According to the literature, we selected nitrilase with high enzymatic activity and relatively complete sequences.

which are NhNIT（BBa\_K3599002）, AdNIT（BBa\_K3599003）,PfNIT(BBa\_K3599005) ，AfNIT(BBa\_K3599006)and LaNIT (BBa\_K3599004). We successfully constructed 3 NITs which are pTac\_NhNIT(BBa\_K3599007）, pTac\_AdNIT（BBa\_K3599009）, and pTac\_LaNIT（BBa\_K3599008） and tested the enzymatic activity of La & AdNITs.

We aimed to find a broad-spectrum aromatic sensor reacting to PAN molecules.We selected candidates sensor by finding the small molecule with the best response and the similarity of the chemical structure with PAN.We designed a PaaX \_eGFP(BBa\_K3599019) and Paax\_sfGFP (BBa\_K3599020).

### pTac\_NhNIT,pTac\_AdNIT,pTac\_LaNIT construction

The functional sequence we got from the company is constructed on pUC57 backbone. But pUC57 is not attached to all of our designs. Therefore, we need to rebuild the plasmid and change the arrangement of sequence. Our effector system is constructed on Pet28-b plasmid and binds with the upstream LacI sequence.

![](/wiki/images/b/b2/T--TPR_China--eng01.jpg)

We verified monoclones by PCR with specific primers seq-28b F/R.

### pTac\_NhNIT(BBa\_K3599007)

![](/wiki/images/f/f9/T--TPR_China--eng02.jpg)

###### Fig.1 The result of pTac\_NhNIT construction

### pTac\_AdNIT(BBa\_K3599009)

![](/wiki/images/4/4b/T--TPR_China--eng03.jpg)

###### Fig.2 The result of pTac\_AdNIT construction

### pTac\_LaNIT(BBa\_K3599008)

![](/wiki/images/5/50/T--TPR_China--eng04.jpg)

###### Fig.3 The result of pTac\_LaNIT construction

According to the design of NIT, the length of pTac\_NhNIT should be 1350bp, the length of pTac\_AdNIT should be 1300bp, and the length of pTac\_LaNIT should be 1350bp. According to the PCR results shown in Fig.1 to Fig.3, we constructed them successfully.

## 1. SDS-PAGE verification of nitrilase

We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.

![](/wiki/images/e/ee/T--TPR_China--eng05.jpg)

###### Fig.4 The SDS-PAGE result of fermentation broth

Electrophoresis results show that our sample has a specific band around 31KD compared to the negative control，this proves that the engineered bacteria we constructed have expressed nitrilase protein.

## 2. Nitrilase Enzyme Activity

The nitrilase has PAN as substrate to degrade PAN into phenylacetic acid and ammonium. Ammonium and Nash reagents can produce reddish-brown precipitation. The shade is proportional to the concentration of NH4+ produced by PAN degradation.

### • Standard curve

We plotted the standard curve of the ammonium concentration.The experimental sample's NH4+ concentration can thus be calculated from the standard concentration equation.

![](/wiki/images/2/25/T--TPR_China--eng06.jpg)

###### Fig.5 Standard curve of NH4+

### • Nitrilase Enzyme Activity

The activity of nitrilase was verified by detecting the ammonium ions produced by the degradation of PAN by the bacterial liquid. We define U as the amount of PAN that can be degraded per microliter per hour.

![](/wiki/images/6/68/T--TPR_China--eng07.jpg)

###### Fig.6 Experimental phenomena of enzyme activity test

![](/wiki/images/3/30/T--TPR_China--eng08.jpg)

###### Fig.7 Enzymatic activity of Nitrilase

As can be seen from Fig. 7, the U value of pTac\_LaNIT is 0.6115, which is 1.27 times that of the negative control;the U value of pTac\_AdNIT is 0.6205, which is 1.3 times that of the negative control.These data indicate that our engineered bacteria have high activity of nitrilase.

## 3. Construction of engineering bacteria for sensor

### • Paax \_eGFP（BBa\_K3599019）,Paax \_sfGFP（BBa\_K3599020）

We aimed to find a broad-spectrum aromatic sensor reacting to PAN molecules.We designed a PaaX Sensor.

![](/wiki/images/5/52/T--TPR_China--eng09.jpg)

###### Fig.8 Biosensor circuit of Paax\_eGFP(BBa\_K3599019）

![](/wiki/images/7/77/T--TPR_China--eng10.jpg)

###### Fig.9 Biosensor circuit of Paax\_sfGFP（BBa\_K3599020）

### • The result of sensor construction

![](/wiki/images/c/cc/T--TPR_China--eng11.jpg)

###### Fig.10 The result of Paax\_-TF construction

![](/wiki/images/7/7a/T--TPR_China--eng12.jpg)

###### Fig.11 The result of Report construction

According to the design of sensor,the length of Paax-TF should be 1200 bp. According to the PCR results shown in Fig.8 to Fig.11, they have been constructed successfully.

## 4. Sensor induced by small molecules The use of sensors can be divided into two steps. First, we used the most sensitive molecule of each sensor that was reported by the previous teams, to test whether the sensor itself is good. After we confirmed that the sensor is good, then it was tested to see whether it responds to the PAN. • The fluorescence of the sensor induced by small molecules ![](/wiki/images/8/81/T--TPR_China--eng13.jpg) Fig.12 The fluorescence of the Paax- sensor induced by small molecules Through the corresponding small molecule sensing experiment, Paax\_eGFP and Paax\_sfGFP have great induction effects on aromatic small molecules. • The fluorescence of the sensor induced by PAN ![](/wiki/images/f/f9/T--TPR_China--eng14.jpg) Fig.13 The fluorescence of the Paxx sensor induced by PAN And here is the fluorescence of different sensors induced by PAN. Through the corresponding small molecule sensing experiment, Paax\_eGFP and Paax\_sfGFP have great induction effects on PAN.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TPR China/Experiments

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### 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://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:TPR China/Results

# Results

You can describe the results of your project and your future plans here.

### 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 also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* 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)