Team:HiZJU-China/Implementation
In this program, we targets on the increasingly severe pollutant Ethinylestradiol and provide a convenient solution for governments and individuals to tackle the issue of detection and dispose. In order to curtail the cost and reduce the time consuming we employ low-cost quantitative and qualitative stratagem on technological process to two sets of application environment. For the user with no biology environment we design a chemical chromogenic method for qualitative detection of EE2, while we construct an integral route on industrial processing, which can automatically diminish the concentration of EE2 within natural watershed.
Our implementation technique are combined with parts below.
For boarder application environment and higher degradation rate, the part of the project introduce knowledge on synthetic biology and bio-chem reaction kinetics embodied in the transfer of amoA and Hao gene from Aeromonas hydrophila to E. Coli. At the same time we optimize the promoter in accordance with the Heterotrophic organisms character. Finally we attained to degrade both EE2 Homologue and nitrogen amid water and bring more prospect to Co-metabolism. In the idea of industrial wastewater treatment, we will use mature sodium alginate embedding method to fix engineering bacteria and make it into multistage biofilm and apply it to biological storage tank, as the terminal device of sewage treatment, to treat EE2 and a certain concentration of ammonium concentration in wastewater economically and efficiently.
Targeting at the defect of temporary detection device like HPLC,time-consuming, cumbersome and expensive, we resort to synthetic biology and two-hybrid yeast technology and import Plasmid coupling protein and receptor into Pichia pastoris. This Recombinant Fluorescent Yeasts can show a linear correlation between Fluorescent lightness and EE2 content. Which obtain the characteristics of low cost, short duration and easy operation. In industrial applications, we designed a completely shading biological detection device to store the fluorescent and by detecting the light emitting from green fluorescent protein activated by LED and use photosensitive resistance to detect fluorescence intensity of wastewater, we can get the excat concentration of EE2 in water by batches. Electric circuit design is also concluded to switch from emission or recycle of the waste. Our analysis method can greatly improve the detection speed, so as to improve the detection efficiency of the relevant departments, expanding the scope of the inspection
Applying bioengineering knowledge, the degradation part and the detection part are combined into a semi-automatic organization that is suitable for large-scale industrial application scenarios supported by the government to deal with estrogen pollution in a certain watershed. The one-way flow filtration mechanism is used to prevent the engineering bacteria in the culture tank, degradation tank and detection point from leaking, and batch sampling is used in the internal cycle processing to realize the integration of degradation, detection and discharge.
For the two separation parts of our industry-oriented effluent treatment mechanism we apply kill switch into our engineered microbe. For the detection part we introduce L-arabinose-operating system to switch on the mazF gene to kill the engineered bacteria when leaking in the out-side environment in lack of L-arabinose. In that way additional regulatory will not be necessary thus curtail the cost. What’s more, it will be a visionary attempt to apply this kill switch to other engineered bacteria.
Fig.1
For the degradation part we accordingly apply two layers structured kill switch to interrelate the concentration of EE2 and the L-arabinose-operating system. The combination of EE2 and ER receptor will inhibit CⅠDN molecules to express and consequently activate the downstream process initiated by PRM promoter leading to the expression of toxic protein mazF.
Fig.2
The next part is the safety secure method in our engineering implementation. We based on the mainstream wastewater processing mechanism and raise its safety-related level presented in the one-way valve to control the flow of air, the Sodium alginate crosslinking method to stabilize the bacteria and fix them on the multi-layer biofilm. We also append a filter at the end of the processing route to realize the aseptic output after the complete flow scheme.
Additionally, we provide those personal-use-only detection pack a non-contact way to detect the EE2 concentration. The two-hybrid yeast will be fixed in the plastic culture dish at bottom of colourimetric card. Then they can mix medium powdery and water to be detected together on appointed volume. The solution can be added through a one-way port and the whole qualitative analysis process will be finished in a insulated environment.
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