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Team:BIT/project biosensors
From 2013.igem.org
| Beta-lactam Detection Device |
| Background |
| In order to prevent cow mastitis, all the producers of diary products feed the cows with antibiotics. However, excessive residual antibiotics will increase the drug resistance on human body. According to international standards for antibiotics, most dairy farmers use beta-lactams, such as penicillin deviants and cephalosporin which exceed quality standards on their cows. The beta-lactam biosensor is designed for the detection of beta-lactam in dairy products. Beta-lactam biosensor is aimed to create a biosensor that can be applied in practical life. It is useful for citizens to know what they drink and what they buy for their little babies are qualified and hygienic. While there are traditional methods to detect beta-lactam antibiotics, such as enzyme-linked immunosorbent assay (ELISA) and ECLIPSE50, all these methods have to rely on laboratories which are equipped with precise instruments. In order to solve the problem, our Beta-lactam biosensor is designed to be used on on-site detection in a few hours by users without special training. |
| Device |
| Beta-Lactam antibiotics have become less effective for the treatment of staphylococcal infections as a result of the bacteria's resistance to Beta-Lactam increases sharply during the past few years. Researches have shown that the resistance is mediated by beta-lactamase (encoded by blaZ) that hydrolyzes penicillin whose transcription is regulated by related regulators (encoded by blaI). The purified repressor(BlaI) of beta-lactamase production has been shown to bind specifically to two regions of dyad symmetry, known as operators, which are located between the divergently transcribed beta-lactamase structural gene(blaZ) and the gene(blaR1) encoding the putative transmembrane sensor protein. The bla operon has been found that is induced by beta-lactam. |
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| Hypothesis identified bla as a beta-lactam-sensing operon of beta-lactamase expression, so we designed two devices working in E.coli (DH5α) to build the beta-lactam biosensor. |
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| This device will work to detect the concentration of Beta-Lactam in dairy products. At the same time, we designed another two devices to decrease the detection limit. |
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| Result |
| We prepared a series of bera-lactam solution of which the concentration was respectively 0 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 5 μg/mL, 100 μg/mL and 200 μg/mL, then we add the solution to the bacteria liquid with BBa_K1058009 of which the OD is around 0.2~0.3 with the ratio of 1:1000 respectively, and the concentration of beta-lactam in the environment of the engineering E.coli in 8 different tubes is respectively 0 ng/mL, 10 ng/mL, 20 ng/mL, 30 ng/mL, 5 ng/mL, 100 ng/mL and 200 ng/mL. The samples were taken to two 96-well plates once per hour or once per 30 minutes. The intensity of green fluorescence was tested with a fluorescence microplate reader. The results are as follows. |
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| Tetracycline Detection Device |
| Device |
| Our project is centered on creating a working toggle switch which changes between two different states when chemicals are added. We started with a simple switch that utilizes two inhibitor proteins, LacI and TetR, which bind to sites on the pLac and pTet promoters, respectively. When bound to the promoter, it would not start transcription and produce the green protein. However, certain chemicals (tetracycline and IPTG) will prevent the inhibitor from binding to their respective promoters. So, the promoters are unlocked and the green fluorescence protein is produced. |
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| When there is no tetracycline, the pTet promoter is locked, which means no fluorescence will be produced. |
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| However, when tetracycline and IPTG are added, the TetR protein combines with the tetracycline. At the same time, the pTet promoter transcripts the the T7polymerase, which binds to the T7 promoter. Because IPTG has activated the LacI, the green fluorescence protein will be produced. Moreover, as the concentration of tetracycline is increasing, the intensity of the fluorescence will increase spontaneously. |
| Result |
| A series of tetracycline solution of different concentration was prepared, then we add the solution to the bacteria liquid with tet sensor of which the OD is around 0.2~0.3 with the ratio of 1:1000 respectively, and then add milk into the mixture with the ratio of 1:9. The samples were taken to two 96-well plates once per hour or once per 30 minutes. The intensity of green fluorescence was tested with a fluorescence microplate reader. The results are as follows. We can tell that the maximum of the fluorescence intensity is at the concentration of 15~20 ng/mL. P.S. The horizental coordinate of the first picture is the concentration of the tet in milk, which of the other pictures is the concentration of the tet in the mixture of milk and bacteria liquid, thus the graphs of the first picture and the others have a relationship of 10 times. |
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