Team:HiZJU-China/Proof Of Concept
We raised synthetic biology means to tackle the contaminant problems, but how did we rooted our idea on the ground?
1. We tested our parts in relevant environment and generated desirable results.
2. We designed different types of hardware to suit various need.
Degradation System based on genetically modified E.coli BL21 was supposed to decrease the content of EE2 with the existence of ammonia or urea. After successfully expressed the target protein amoA and HAO in engineered bacteria (NB1) , we incubated NB1 with 10mg/ml ammonia, 1.5μg/ml EE2 (50ml system)at 28℃ for 48h, then we detected the EE2 concentration by HPLC-hv while determined the ammonia concentration by ELIASA. As to urea, we chose the initial concentration of 40mg/ml and also detected it by ELIASA.
Fig 1 the change of concentration of ammonia with time
Fig 2 the change of content of nitrite ion with time
Fig 3 the ability to degrade urea
The content of EE2 in the medium was determined by HPLC. The amount of EE2 was represented by peak area. We found that compared with the blank control, the transformed bacteria could degrade EE2 (Fig. 4).
Fig 4 the standard curve of EE2
Fig. 5 Relationship between the peak area of EE2 and culture time.
Fig 1 the change of concentration of ammonia with time
Fig 2 the change of content of nitrite ion with time
Fig 3 the ability to degrade urea
Fig 4 the standard curve of EE2
Fig. 5 Relationship between the peak area of EE2 and culture time.
At present, the detection of ethinylestradiol (EE2) mostly rely on expensive and complex instruments, such as high performance liquid chromatography(HPLC). Besides, detection also need to be operated by professional people. These reasons make detection of estrogen high-cost, time-consuming and lack of portability. With the increasing awareness of the environmental pollutant, estrogen, the existing detection methods obviously can not meet the increasing requirement of detection.
In order to solve the above problems and meet the needs of a various demand of estrogen detection, we combined the engineering bacteria with the diversified engineering needs. There are three specifications of detection devices: Civil detection, High throughput 96-well plates testing equipment for laboratory, and estrogen degradation model of sewage treatment plant.
The hardware design of civil detection is mainly characterized by "simplicity" and "portability". The whole hardware consists of three parts: freeze-dried engineering bacterial powder, culture medium and sample adding tank. The civil version use the red pigment expressed by Y2H yeast itself, which is positively correlated with estrogen concentration in a certain range. Therefore, the final result will be expressed in different degree of red. Compared with reference color card, the final sample concentration can be semi-quantitatively determined. The operation is simple and sensitive. There are two partitions in the whole kit. The partition of the second layer and the third layer contain the air, culture medium and yeast powder. Pull partition out before use to make the yeast fully contact with the culture medium and air. The engineered bacteria were resuscitated for a certain period of time until their activity was sufficient to complete the following detection tasks. Then, the engineering bacteria are mixed with the sample, and the concentration of estrogen in the sample can be quantified by observing the mcherry produced by the reaction.
The high-throughput 96-well plates used in the laboratory are mainly used for the detection of fluorescence expressed by Y2H yeast, supplemented by flow cytometer or photosensitive circuit for rapid and high-throughput detection. Based on the principle of fluorescence quantification, our device can detect the estrogen content in liquid samples. The method of detection has the characteristics of simple operation, reacting quickly and no need for a large number of samples. The detection of fluorescence makes the experimental results more accurate, and the characteristics of high throughput allow the experimenter to detect multiple samples at one time. To eliminate interferences from the external environment, the detection room is a complete dark container. We use light- emitting diodes (LED) that emit a specific wavelength (395nm) and detect the fluorescence intensity at 509nm, then quantify the fluorescence intensity by converting it to a physical quantity (current/voltage) that is easy to measure.
The estrogen degradation model of sewage treatment plant takes the degradation tank as the main part, supplemented by detection equipment. If the sample is lower than the concentration requirements, it can be discharged. If it is higher than the concentration requirements, it will enter the circulating pipeline for secondary degradation.
It is mainly divided into two modules: degradation module and detection module.
The degradation module is composed of a culture tank for expanded culture of degradation engineering bacteria and a degradation tank for elimination estrogen in sewage.
The detection module is composed of sampling point, detection engineering bacteria culture tank, optical detection equipment and signal processing system.
Judge whether the sewage sample meets the standard through optical detection equipment and signal processing system.
At present, the detection of ethinylestradiol (EE2) mostly rely on expensive and complex instruments, such as high performance liquid chromatography(HPLC). Besides, detection also need to be operated by professional people. These reasons make detection of estrogen high-cost, time-consuming and lack of portability. With the increasing awareness of the environmental pollutant, estrogen, the existing detection methods obviously can not meet the increasing requirement of detection.
In order to solve the above problems and meet the needs of a various demand of estrogen detection, we combined the engineering bacteria with the diversified engineering needs. There are three specifications of detection devices: Civil detection, High throughput 96-well plates testing equipment for laboratory, and estrogen degradation model of sewage treatment plant.
The hardware design of civil detection is mainly characterized by "simplicity" and "portability". The whole hardware consists of three parts: freeze-dried engineering bacterial powder, culture medium and sample adding tank. The civil version use the red pigment expressed by Y2H yeast itself, which is positively correlated with estrogen concentration in a certain range. Therefore, the final result will be expressed in different degree of red. Compared with reference color card, the final sample concentration can be semi-quantitatively determined. The operation is simple and sensitive. There are two partitions in the whole kit. The partition of the second layer and the third layer contain the air, culture medium and yeast powder. Pull partition out before use to make the yeast fully contact with the culture medium and air. The engineered bacteria were resuscitated for a certain period of time until their activity was sufficient to complete the following detection tasks. Then, the engineering bacteria are mixed with the sample, and the concentration of estrogen in the sample can be quantified by observing the mcherry produced by the reaction.
The high-throughput 96-well plates used in the laboratory are mainly used for the detection of fluorescence expressed by Y2H yeast, supplemented by flow cytometer or photosensitive circuit for rapid and high-throughput detection. Based on the principle of fluorescence quantification, our device can detect the estrogen content in liquid samples. The method of detection has the characteristics of simple operation, reacting quickly and no need for a large number of samples. The detection of fluorescence makes the experimental results more accurate, and the characteristics of high throughput allow the experimenter to detect multiple samples at one time. To eliminate interferences from the external environment, the detection room is a complete dark container. We use light- emitting diodes (LED) that emit a specific wavelength (395nm) and detect the fluorescence intensity at 509nm, then quantify the fluorescence intensity by converting it to a physical quantity (current/voltage) that is easy to measure.
The estrogen degradation model of sewage treatment plant takes the degradation tank as the main part, supplemented by detection equipment. If the sample is lower than the concentration requirements, it can be discharged. If it is higher than the concentration requirements, it will enter the circulating pipeline for secondary degradation.
It is mainly divided into two modules: degradation module and detection module.
The degradation module is composed of a culture tank for expanded culture of degradation engineering bacteria and a degradation tank for elimination estrogen in sewage.
The detection module is composed of sampling point, detection engineering bacteria culture tank, optical detection equipment and signal processing system.
Judge whether the sewage sample meets the standard through optical detection equipment and signal processing system.
