Team:Shanghai city/Design
Design of Our Project
Detection of Agricultural Chemical Pollution
Overview
Agricultural pollution means pollution of the environment and related surroundings caused by using natural and chemical products in agriculture. The pollution sources are harmful to all the living organisms that obtain energy from the food cultivated on farms. Agricultural pollution tends to be more serious since the demand for food is increasing, proportional to the increase in population.
Atrazine, which is produced by an agrochemical company named Syngenta in Swiss, was first registered for use as an agricultural herbicide in the United States in 1959. It is widely used by farmers to control broadleaf weeds and grasses that disturb the growth of corn, sorghum, sugar cane, and other crops. It is also used as a kind of weed killer on golf courses as well as a variety of commercial and residential lawns.80 million pounds of the stuff is used each year in the United States and it is now the second most used herbicide in the US after glyphosate.
Numerous studies have provided evidence concerning the problems caused by atrazine such as increased risk of prostate cancer, decreased sperm count in men, and a higher risk of breast cancer in women.
Declines of endangered amphibians and many other endangered species throughout the country are also linked with Atrazine. It is an endocrine disruptor that directly affects the sexual development of amphibians by changing their hormone cycle.
The goals of our project
Since the methods used presently for detection such as Gas chromatography-mass spectrometry (GC-MS) and Liquid chromatography-mass spectrometry (LC-MS) are high costs, not convenient to carry and hard to operate. So, we want to establish a simple and convenient method for Atrazine detection by Synthetic Biology.
Process
Mechanism of Atrazine biosensor
Some Transcription factors can bind to specific small molecular compounds to activate specific cognate promoters, thus activating downstream reporter expression.
Step I: Atrazine is converted to cyanuric acid by three enzymes AtzA, AtzB, and AtzC at the atrazine degradation pathway.
Step II: After the combination of cyanuric acid and specific transcription factor AtzR, promoter PatzD was activated, thus activating GFP expression.
Flow
Construction of plasmid
Cyanuric acid sensing function test
Construction of Plasmid
We want to construct 4 different plasmids: pME- atzABC, pME-atzR, pME-atzABCR and pBBR-PatzD-GFP for degradation of atrazine, atrazine biosensor and cyanuric biosensor initially. Finally, pME-atzR and pBBR-PatzD-GFP are constructed to test the cyanuric biosensor since some experiments can’t be done during the training.
PatzD is cloned using Citronellanol pseudomonas as template; GFP is cloned using pUC57-GFP plasmid as template; T7-terminator is cloned usingpET-28a as template;
AtzR is cloned using Citronellanol pseudomonas as template; pBBR1MCS-2 is linearized through PCR using pBBR1MCS-2 as template. PatzD- GFP- T7 terminator are overlapped using PCR.
Plasmid pME6032 and pBBR1MCS-2 are extracted from E. coli using Tiangen plasmid extraction kit. pME6032 and AtzR are digested by restriction endonuclease KpnI and XhoI, then connected using T4 NDA ligase. pBBR1MCS-2 and PatzD- GFP- T7 terminator are digested by restriction endonuclease EcoRI and BamHI, then connected using T4 NDA ligase.
During the experiments, we had problems with overlap PCR since the two gene fragments were a bit of too long to combine. Trying the same experiments using different concentration and calculated the adding proportion for several times, our team finally succeed in overlap PCR.
Cyanuric Acid Sensing Function Test
These two constructions are transformed into Pseudomonas putida competence cell by electroshock and cultured in LB medium with kanamycin and tetracycline antibiotics. Then functional test is done.
Core
Skills
We mastered experimental skills in molecular biology such as plasmid extraction, PCR, agarose gel electrophoresis, make receptive state, heat shock transform competence cell, electroshock transform competence cell, making LB medium. Besides, we also mastered how to use machines.
Future Plan
We should continue to construct another vector for atrazine degradation test and atrazine biosensor functional test to confirm the practicability in reality.
There are more details to explore: Can Pseudomonas putida with BD plasmid detect a small dose of atrazine pollution in water?
Our subject is undoubtedly very novel since there are no much similar researches about it, so we can continue the research.
Reference
[1] Regulation of the atrazine-degradative genes in Pseudomonas sp. strain ADP
[2] 节杆菌AD26的分离鉴定及其假单细胞菌ADP对阿特拉津的联合降解
[3] Design and Optimization of a Cell-Free Atrazine Biosensor
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