Technical Research On High-throughput And Rapid Screening Of Environmental Estrogens By Genetic Engineering Bacteria

In recent years, the pollution of the environmental estrogens and its impact on human health have been paid more and more attention to. But, determination technique at present is too complex and time-consuming with expensive instruments. In this research, a genetic engineering bacteria has been constructed and it is sensitive to environmental estrogens. Due to the insert of estrogen-sensitive intein gene into certain color protein gene, when it meets the estrogens in environment samples, the induced cleavage of intein will produce active color protein and therefore the bacteria become colored so that the qualitative detection could be achieved. At the same time, the activity of color protein was positively correlated with the content of estrogen and the quantitative analysis could be realized.With this strain the estrogenic activity of 17β-E2 was detected. Concentration-activity curve of the 17β-E2 shows an S shape and its detection limit is at the level of ng, which means that the strain is sensitive to esrogenic chemicals and could be used to detect chemicals possess estrogenic activity. Also bisphenol A (BPA) was detected and the results show significant dose-response relationship. With this strain, the estrogenic activity of three kinds of polycyclic aromatic hydrocarbons (PAHs), including chrysene, pyrene and benzo (a) anthracene was detected. Results showed strong estrogenic activity for chrysene and nearly no estrogenic activity for pyrene and benzo (a) anthracene, which might be relative to their structures. In order to verify the role of substituents, the 6OH-Chrysene was used and the result showed a significant dose-response relationship, conformed again a stronger relationship of estrogen activity with spatial structure and indicating weaker relationship with the substituent.Molecular docking method has been used to analyze the relationship between the structure of PAHs and the estrogenic activity. The results showed that molecular spatial structure is indeed related to estrogenic activity and the chemicals with closer spatial structure to 17β-estradiol present stronger estrogenic activity.The estrogenic activities in the samples extracted from wastewater and sludge of a sewage treatment plant were detected, and compared to the concentrations of synthetic musk determined by the GC/MS method. The estrogenic activities of the extractants were correlated positively with the GC/MS results, indicated this assay could quantify estrogenic risk on environmental samples quickly and efficiently.In summary, the advantage of the technique with gene engineering bacterial includes fast, simple, inexpensive, highly stable, sensitive and the lowest detection limit can be at ng level. So that this method is particularly suitable for fast scanning of large numbers of samples. It is helpful for ecological risk assessment and provides technical support for the optimization of the water pollution control technique.