Team:ECNUAS/Engineering
Engineering
Background
Atrazine is a herbicide compound commonly found in agriculture. It can diffuse into
soil and water. Atrazine can be degraded into cyanuric acid in this process, which leads to the accumulation
of cyanuric acid in the environment, and has harmful effects on all organisms in the environment. Although
Atrazine is currently banned in most European countries, the herbicide is still widely used in other parts
of the world. Atrazine in the environment can be monitored through various chemical analyses, including gas
chromatography and some physical and chemical detection systems. Although these methods can sensitively
measure the atrazine content in the environment, the steps are cumbersome, expensive, and time-consuming,
and require extensive infrastructure and technical expertise.
Design
Biosensor consisting of a cell-free systems that are sensitive and specific to
specific analytes. It can directly detect the atrazine in sample. Cell-free atrazine biosensor consists of
three to four components, which can promote gene expression and metabolism in vitro (Figure 1). (1) cell
lysate/extract, which contains the cellular mechanisms required for protein synthesis; (2) buffer mixture of
phosphorylated energy substrates, nucleoside triphosphates (NTP), amino acids, salts and other necessary
cellular cofactors; (3) DNA templates that define the genetic program to be performed in the reaction; and
the reaction Any other optional exogenous cofactors, substrates or inducers required. The biosensor detect
cyanuric acid (CYA), a degradation product of atrazine. After atzR binds to CYA, it activates the promoter
Provoin5, thereby activating downstream genes and inducing the expression of the reporter gene GFP.
Figure 1. Composition of the cell-free atrazine biosensor.
Build
Figure 2. Schematic maps of Ptat_AtzR (sensor plasmid) and Pprovoin5_amilGFP (reporter
plasmid).
Test
After the ice-breaking game, we played a short playlet.
We invited the children to take part in the drama. In the relaxed and humorous storyline, we gave the children a preliminary introduction to the dangers of atrazine, and led them to our theme -- what do we know about atrazine?
We invited the children to take part in the drama. In the relaxed and humorous storyline, we gave the children a preliminary introduction to the dangers of atrazine, and led them to our theme -- what do we know about atrazine?
Figure 3. Histogram of the fluorescence intensity 4 hours after 30 μM CYA was added in the
culture.
In order to analyze the relationship between the concentration of cyanuric acid and
the fluorescence intensity, we designed the control groups and collected the data as showing above.
According to the histograms (Figure 4), the fluorescence intensity shows a decreasing trend with the
increase of concentration of cyanuric acid when we used the bacteria C for tests.
Figure 4. Histogram of the fluorescence intensity of bacteria C after 4 and 6 hours under different
concentration of the cyanuric acid.
Learn
In this project, we successfully atrazine biosensor in bacteria. However, cyanuric
acid might affect the growth of strains so that the higher the concentration of the cyanuric acid, the worse
the growth of the bacteria, the less of the amount of the effective “biosensor”. In order to fully eliminate
this impact, we introduced the concept of cell-free extraction and cell-free expression in the next stage of
our project.