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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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| Device 1 |
| 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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| Device 2 |
| 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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| Chromate Detection Device |
| Background |
| Some illegal dairies always add leather hydrolysate into fresh milk and powdered milk to increase the percentage of protein in milk. Chromate, which is one of the elements of leather dye, is the main element that can be used to trace leather hydrolysate. Our Cr(VI)-biosensor is thus designed for the detection of chromate in dairy products. Our Cr(VI)-biosensor is designed to work in places where traditional biosensors cannot. This is important for consumers to know that what they buy for their consumption is qualified and safe to drink. While there are traditional methods for detection of chromate(such as Graphite furnace atomic absorption method, Oscillographic polarography, ICP-AES, High performance liquid chromatography, Spectrophotometric investigation,etc.), all these methods have to rely on laboratories equipped with precise, expensive, experimental apparatuses. However, with our Cr(VI)-biosensor, even consumers without specific training will be able to use it and the results will be knownin just a few hours. Cr(VI) is one of the major environmental contaminants, which reflects its numerous high-volume industrial applications and poor environmental practices in the disposal of chromium-containing waste products. High solubility and tetrahedral conformation of the chromate anion promote its rapid transport across biological membranes, and once internalized by cells, Cr(VI) exhibits a variety of toxic, mutagenic, and carcinogenic effects. Chromate and sulfate are structurally similar anions, which makes it difficult for cells to differentiate between them and is the basis for cellular uptake of chromate by sulfate transporters. Formation of DNA damage is a major cause of toxic and mutagenic responses in both human and bacterial cells, as evidenced by their increased sensitivity to chromate in the absence of DNA repair. Human and other mammalian cells lack detectable extrusion of chromate, and DNA repair is their main cellular defense mechanism against chromate toxicity. Because bacterial cells are less proficient in repair of chromium-DNA adducts compared to human cells, their ability to survive in the environment with heavy chromate contamination requires selection of alternative resistance mechanisms. |
| Design |
| Genes conferring resistance to chromate have been found in Pseudomonas spp., Streptococcus lactis, Ochrobactrumtritici 5bvl1 and Cupriavidusmetallidurans. The 7,189-bp-long TnOtChr of Ochrobactrumtritici 5bvl1 contains a group of chrB, chrA, chrC, and chrF genes situated between divergently transcribed resolvase and transposase genes. |
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| The chr promoter was strongly induced by chromate or dichromate, but it was completely unresponsive to Cr(III), oxidants, sulfate, or other oxyanions. Plasmid reporter experiments identified ChrB as a chromate-sensing regulator of chr expression. According to this evidence, we designed three kinds of devices working in E.coli (DH5α) to build Cr(VI)-biosensor. |
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| The chr promoter has a weak constitutive expression without chromate, while it is strongly induced to express. This device will work to detect the concentration of chromate in dairy products. At the same time, we designed another two devices to reduce the detection limit. |
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| Device 1 |
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| Device 2 |
