Team:Shanghai high school/Design
Design
Overview:
Environmental deposition of natural and synthetic chemicals with estrogenic properties is associated with numerous human and wildlife physiological disorders, leading to the development of various estrogenic chemical screening methods. Conventional methods, such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC), are accurate and have been widely used in estrogenic chemical detection. However, as estrogenic chemicals have diverse structures and properties, conventional methods aren’t suitable for quick detection. However, the expensive detection instruments and professional operation of detection make the above methods difficult to be widely used in agriculture and environmental detection, especially for individual users or quick detection applications.
Genetically coded biosensors have been widely used in the detection of chemicals in recent years. These biosensors can specifically transform a chemical into color, fluorescence, or other detectable signals, which have the characteristics of low cost and easy operation. Among them, protein biosensor has the advantages of sensitive response and fast detection speed, while whole-cell biosensor has the characteristics of wide application range and low cost.
An intein is a protein element found as an in-frame insertion within the sequence of a particular host gene. It can cut itself post-translationally from host protein and ligate adjacent peptide sequences with peptide bonds via self-catalyzed protein splicing. In general, inteins’ insertion into host protein inactivates its activity, and only after protein splicing can the host protein’s activity be restored. Most interins have two domains, one splicing domain and one endonuclease domain. In short, inteins are commonly used as switches in the regulation of protein activities. Inspired by the splicing function of inteins, in this project, we are trying to use the engineered bacteria to detect the pollution of the environmental estrogens. The engineered bacteria will express a chimeric sensor protein which consists of an estrogen-sensitive intein and a reporter protein lacZ. The chimeric sensor protein will produce active lacZ that can be detected only when contact with estrogens in environmental samples.
Expectation:
We want to use our engineering bacteria to detect estrogenic chemicals in environmental water samples by expressing a chimeric sensor protein, which is constructed by the insertion of an estrogen-sensitive intein part into lacZ. The estrogen-sensitive intein also named VMAER inteimposed of two regions: the splicing region of VMA intein and the human estrogen receptor gene. The chimeric sensor protein in this project can be divided into five parts: N-terminus of LacZ gene (a reporter gene), N-terminus of VMA intein, human estrogen receptor coding sequence, C-terminus of VMA intein, and C-terminus of LacZ gene.
The sensor protein will be expressed in E. coli and obtained by cell lysis as the detection system. In the presence of estrogen or its analogs, the VMAER intein splices itself out from the LacZ protein efficiently, and the active LacZ protein is produced to generate positive results in the -galactosidase assay: After the addition of X-gal, the solution turns blue. Otherwise, no galactosidase activity can be detected and the detection system remains to be colorless.
Process:
1. Linearize plasmid vector via restriction enzyme digestion, use overlapping PCR to create our target DNA sequence.
2. Design primers that contain homologous sequences to amplify sequence 12345, which allows recombination of target DNA sequence into pET-28a.
3. Linearized plasmid vector and the inserted target sequence were mixed at a certain molar ratio (usually 1:2), allowing homologous recombinant reaction to be completed. The expected recombinant vector is formed.
4. Transform the recombinant vector into competent cells (DH5-alpha): Hundreds of monoclonal plasmids are formed in petri dishes; In later stages, antibiotic resistance and genetic sequencing were used for identifying successfully transformed DH5-alpha cells.
5. Use colony PCR to determine whether or not our desired recombinant vector proliferates.
6. Extract recombinant vectors and run gel-electrophoresis to determine the size of vectors present in DH5-alpha and therefore evaluate how successful recombination is.
7. Transform recombinant pET-28a into BL21 cells.
8. In vivo experiment: inducer IPTG and X-Gal were added to the bacterial solution on culture dishes to see if the solution changes color under different concentrations of estrogen.
9. In vitro, the purified proteins extracted from BL21 bacterial cell solution were directly reacted with X-Gal in a water bath to observe the color change. Purified proteins are also tested using protein electrophoresis.
Principle explanation:
1. To construct the recombinant plasmid, the following steps are required:
I. Primer design
II. Select appropriate vectors, cleavage sites.
III.Confirm the base sequence of the target DNA sequences on NCBI.
IV. Submit toa company for primer production
Overlapping PCR→ Restriction enzyme digestion, ligation→ Transformation→ Plasmid extraction→ Collect→ Resuspend→ Lysis→ Wash→ protein dissolution→ protein electrophoresis.
Reference：
1. Chong, S., and M. Q. Xu. 1997. Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J. Biol. Chem. 272: 15587–15590.
2. Perler, F. B. 2006. Protein splicing mechanisms and applications. IUBMB Life 58:63
3. Zacharewski, T. 1997. In vitro bioassays for assessing estrogenic substances. Environ. Sci. Technol. 31:613–623.
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