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Revision as of 18:29, 9 November 2020

Antea-Glyphosate
Presented by Team XMU-China 2020

Jinzhu Mao¹, Shi Zhang¹, Siru Zhou¹, Meihao Ou¹, Qingliu Wang¹, Ruijie Mo¹, Ruomeng Bi¹, Shichen Geng¹, Shuling Xiao¹, Xiaoyu Wang¹, Yangqi Deng¹, YiXian Zheng¹, Zhichun Yang¹, Shengyang Zhang¹, Jisheng Xie¹, Yuan Li¹, Gezhi Xiao², Xiao-yan Zhuang², Ai-hui Zhang², Wang Yali², Fu yousi², Junhong Chen², Zinuo Huang², Yang Liang², Xingyu Chen², YanSong Chen², Fude Chen², Shiyi Zhai², Langxing Liao², Limin Qiu², Tianyu Feng², Wenyao Shao², Yajuan Peng², Haitao Wang², Yang Shi², Jiacheng Huang², Baishan Fang³, Xiamen University, Xiamen, Fujian, China

Attributions

Brainstorms: All team members.

Molecular cloning and circuit construction: All team members.

Characterization: All team members.

Proof of Concept: All team members.

Model: Yixian Zheng, Siru Zhou, Ruomeng Bi, Ruijie Mo, Jinzhu Mao.

Human practices: All team members.

Wiki: Shengyang Zhang.

Art design: Yangqi Deng, Fude Chen, Jisheng Xie, Junhong Chen.

!!!这里他妈的缺一张图
Abstract
Tea is deeply rooted in Chinese culture. For a long period, a large amount of glyphosate has been used as a herbicide, which raises a severe problem of pesticide residues in tea and tea products.
!!!这里他妈的缺一张图
This year XMU-China aims at developing an efficient glyphosate detection and degradation system.

There are two pathways found to degrade glyphosate:
►Glyphosate generates AMPA and glyoxylic acid through the C-N cleavage.
The product, glyoxylic acid, could be further converted into measurable signal, thus for the detection system, glyphosate is degraded by several enzymes through C-N cleavage and then transformed into a measurable fluorescence signal caused by the NADPH.
►Glyphosate generates sarcosine and phosphate acid through the C-P cleavage.
The product by C-P cleavage pathway, sarcosine, could be rapidly oxidized to become formaldehyde, a non-toxic substance. Therefore, glyphosate degradation method through C-P cleavage is preferred in our design which plans to disintegrate glyphosate to minimize the toxicity.
!!!这里他妈的缺一张图
►Two kill switches controlled by different inducers are also proposed.
!!!这里他妈的缺一张图
It is hoped that this project could provide new ideas for the detection and degradation of pesticide residues. Taking care of the earth by tiny bacteria, we here promise a better future of tea.
!!!这里他妈的缺一张图
Motivation
We identified 3 areas of synthetic biology that we wanted to improve for the iGEM community: Building, Characterizing, and Sharing.


BUILDING: BioBricks assembly requires multiple cycles of ligations and digestions, making it very time consuming when constructing large circuits

CHARACTERIZING: Characterization methods for circuits containing fluorescent proteins vary, making data comparison challenging

SHARING: Parts pages on the Registry often lack a standard format in which characterization information is displayed
Building: MoClo Assembly Method
Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together.

This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below).

There are three Levels of MoClo Parts (shown below):

Level 0: Basic part (ex: promoter, RBS, CDS, etc.)
Level 1: Transcriptional unit (up to 6 Level 0 Parts)
Level 2: Composite of up to 6 Level 1 parts



Reference: Weber et al., PLoS One 2011 
Building: MoClo Library
We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.

For BioBrick parts >100bp, we used a standard PCR methodology.
We used a ligation PCR methodology for sequences < 100bp in length (J. Lee et al., Biotechniques. 2004).

All parts listed in Table 1 were confirmed with sequencing

All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011).




New Parts
We have cloned a new copper sensitive σ54-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009).

Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned.
Motivation
We identified 3 areas of synthetic biology that we wanted to improve for the iGEM community: Building, Characterizing, and Sharing.


BUILDING: BioBricks assembly requires multiple cycles of ligations and digestions, making it very time consuming when constructing large circuits

CHARACTERIZING: Characterization methods for circuits containing fluorescent proteins vary, making data comparison challenging

SHARING: Parts pages on the Registry often lack a standard format in which characterization information is displayed
Building: MoClo Assembly Method
Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together.

This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below).

There are three Levels of MoClo Parts (shown below):

Level 0: Basic part (ex: promoter, RBS, CDS, etc.)
Level 1: Transcriptional unit (up to 6 Level 0 Parts)
Level 2: Composite of up to 6 Level 1 parts



Reference: Weber et al., PLoS One 2011 
Building: MoClo Library
We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.

For BioBrick parts >100bp, we used a standard PCR methodology.
We used a ligation PCR methodology for sequences < 100bp in length (J. Lee et al., Biotechniques. 2004).

All parts listed in Table 1 were confirmed with sequencing

All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011).




New Parts
We have cloned a new copper sensitive σ54-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009).

Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned.
Motivation
We identified 3 areas of synthetic biology that we wanted to improve for the iGEM community: Building, Characterizing, and Sharing.


BUILDING: BioBricks assembly requires multiple cycles of ligations and digestions, making it very time consuming when constructing large circuits

CHARACTERIZING: Characterization methods for circuits containing fluorescent proteins vary, making data comparison challenging

SHARING: Parts pages on the Registry often lack a standard format in which characterization information is displayed
Building: MoClo Assembly Method
Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together.

This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below).

There are three Levels of MoClo Parts (shown below):

Level 0: Basic part (ex: promoter, RBS, CDS, etc.)
Level 1: Transcriptional unit (up to 6 Level 0 Parts)
Level 2: Composite of up to 6 Level 1 parts



Reference: Weber et al., PLoS One 2011 
Building: MoClo Library
We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.

For BioBrick parts >100bp, we used a standard PCR methodology.
We used a ligation PCR methodology for sequences < 100bp in length (J. Lee et al., Biotechniques. 2004).

All parts listed in Table 1 were confirmed with sequencing

All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011).




New Parts
We have cloned a new copper sensitive σ54-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009).

Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned.
Acknowledgements and Sponsors
Acknowledgements
Dr. Swapnil Bhatia
Janoo Fernandes
Dr. Michael Smanski
Dr. Ernst Oberortner
Jenhan Tao
Dr. Alexy Vorobev


Sponsors