Team:Shanghai high school/Description
Description
Background
In every place and every time in this world, a new life is born, accompanied by a crisp cry. Countless warm families have been formed. They look forward to a bright future for their family and look forward to their children becoming promising people in the future. What they don't know is that a hormone is secretly threatening the healthy development of their children.
Environmental hormones, also called endocrine disruptors, are a kind of chemical additives that are ubiquitous around people and contribute to industrial production. They are abundantly present in cosmetics, pesticides, stimulating hormones, and industrial production. Their widespread presence has brought huge security risks to this society. Bear the brunt of the two most vulnerable groups: infants and pregnant women.
Since these two groups are quite different, we will separately explain the harm of environmental hormones to him.
For infants: excessive exposure to environmental hormones can cause problems in fetal brain development. The brain of a baby boy may be feminine. And in the future work, the work efficiency and memory of babies who are overexposed to environmental hormones will be much lower than healthy babies who have not been exposed to environmental hormones.
For pregnant women: excessive exposure to environmental hormones during pregnancy may cause miscarriage and fetal malformations. Excessive exposure to environmental hormones in pregnant women can cause infertility.
Inspiration
Environmental hormones, or endocrine interferons (EDCs), are a class of compounds that affect human metabolism. These chemicals can mimic natural hormones or hinder the action of normal human hormones. In the past few decades, EDC has proliferated in the environment. Harmful substances have been detected from rivers and the ocean in the world. More than 30 types of endocrine hormones were detected from the seeping water at the industrial waste disposal site. The common types of EDCs are drugs (steroids, acetaminophen), insecticides (DDT), industrial products (bisphenol A, polychlorinated biphenyls), etc. The large-scale application or discharge of these chemicals into the environment has been proved to cause harm to humans and wildlife. For example, bisphenol A (BPA) has been used since the 1960s to make plastic (milk) bottles or suckling cups for toddlers, as well as the inner coating of food and beverage (milk powder) cans. Every year, 27 million tons of BPA containing plastics are produced worldwide. But BPA can lead to endocrine disorders, threatening the health of the fetus and children. Cancer and obesity caused by metabolic disorders are also thought to be related. In addition to the potential harm to humans, endocrine interactions have been shown to affect the growth of wild animals, especially amphibians, resulting in deformity and death. The European Union believes that bottles containing bisphenol A can induce precocious puberty and banned the production of baby bottles containing bisphenol A since March 2, 2011. While estrogens at pollutant levels have been linked with breast cancer in women and prostate cancer in men. Estrogens also perturb fish physiology and can affect reproductive development in both domestic and wild animals.
The common detection methods of the environmental hormones are gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary detection (CE), or enzyme-linked immunosorbent assay (ELISA). But in some cases, it is not suitable to use this expensive instrument in professional laboratories.
As being described above, we hope to develop a sensitive and easy-to-use environmental hormone detection method to facilitate the detection of natural water or agricultural water. Biosensors have been widely used in the detection of compounds in recent years. This method is not only specific, simple but also highly sensitive. We believe that it has the potential to be a new method for portable or household environmental hormone detection.
Description
In this project, we plan to design a protein biosensor for estrogen detection. Protein biosensors can be induced to be allosteric by the detected compounds thus new structural confirmations or new cleavage proteins are produced. This new conformation or cleavage protein usually function as a reporter protein to generate detectable signals, such as fluorescence or color reaction. Compared with the biosensor based on reporter gene expression, protein biosensor has the advantage of faster detection speed.
The key components in our experiments are estrogen sensing / binding protein, a protein that can lead to allosteric/shear, and reporter protein.
Therefore, through the references, we chose the structure of intein for the construction of estrogen biosensor protein. Intein can produce new active proteins by excising itself and join the remaining portions with a peptide bond in a process termed protein splicing. In general, inteins' insertion into the host protein can inactivate the host protein, and only after protein splicing can the host protein's activity be restored. Human estrogen receptor can be used in the estrogen sensing / binding domain of sensor protein, which has been proved to induce the splicing and ligation of the intein under the condition of estrogen binding. Finally, we chose lacZ as the reporter protein. LacZ is a common chromogenic reporter protein, which can react with X-gal in blue or with ONPG in yellow.
In our project, we hope to construct a complete estrogen biosensor protein by inserting the estrogen sensing/binding domain and intein domain into the lacZ protein. In the presence of estrogen molecules, the estrogen sensing / binding domain binds to estrogen and induces the cleavage and ligation of the intein, thus producing a complete and active lacZ protein, which can be detected by the color reaction.
Reference
Skretas G, Wood D W. Regulation of protein activity with small-molecule-controlled inteins [J]. Protn ence: A Publication of the Protn Society, 2005, 14(2).
Markey C M, Rubin B S, Soto A M, et al. Endocrine disruptors: from Wingspread to environmental developmental biology[J]. J.steroid Biochem.mol.biol, 2002, 83(1):235-244.
David W. Wood, Georgios Skretas. Intein Reporter and Selection Systems[J]. Nuclc Acids & Molecular Biology, 1970, 16:325-344.
Buskirk AR, Ong YC, Gartner ZJ, Liu DR. Directed evolution of ligand dependence: small-molecule-activated protein splicing. Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10505-10. doi: 10.1073/pnas.0402762101. Epub 2004 Jul 9. PMID: 15247421; PMCID: PMC489967.
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