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.
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.
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.
The project of Chromate
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.
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.
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.
Device 1
Device 2
Result
Amplifier
If we give the biosensor an input signal, we will get an output signal, which, however, may not be strong enough for us to detect. Therefore, we have designed an amplifier, which is based on the high activity of T7 promoter, to increase the intensity of the output signal to a specific magnification. We replaced the sequence of the green fluorescent protein of the sensor with the DNA of T7 RNA polymerase to promote the expression of the downstream DNA. Thus we can get stronger fluorescent intensity as expected.
As we all know, if the sample of material which the concentration is high enough to be detected is inserted as the input signal, the sensor will be able to "feel" it and produce an output signal.
However, sometimes, the output is not strong enough for us to detect. To magnify the output signal, we took advantage of the high activity of the T7 promoter. Because the T7 promoter can only be activated by the T7 RNA polymerase, a gene of T7 RNA polymerase and a T7 promoter were inserted at the downstream site of the sensor to get a stronger output. This part is what we call an "amplifier".
Controller
Sometimes we need to enhance the output signal to different degrees. In other words, we want to control the magnification. A "controller" is designed to solve this problem. We inserted a lacO operator between the DNA of T7 RNA polymerase and green fluorescent protein, and added a lacI biobrick in the system. When there is low concentration of IPTG, the lacI will close the lacO to inhibit the expression of gfp DNA. When we add IPTG to the sample, the lacI will be combined with IPTG, and the inhibition of the expression of the downstream DNA will be inhibited. Thus we can control the magnification by controlling the concentration of IPTG.
Here we introduced a new part which contains lacI and lacO in the system. The gene of lacI is always expressing, which inhibits the expression of lacO. In this case, even if there is an input signal, no egfp will be expressed.
If we put IPTG in the environment as an inducer, the lacI protein will combine to the IPTG molecules and thus the inhibition will be ceased. As a result, the lacO will be activated, which will lead to the expression of downstream egfp.
With a stable concentration of IPTG, the system will work as expected. When a weak input is given, a weak green fluorescence will be detected, while if the input gets stronger, the intensity of the green fluorescence will increase simultaneously.
Sometimes we would like to control the magnification. This could be realized by regulating the concentration of IPTG. The higher the concentration of IPTG is, the more lacI will be combined. As a result, the expression of downstream egfp will be enhanced.
Similarly, we can decrease the magnification by lowering the concentration of IPTG.
This is the part what we call a "controller".
Hardware
As the joint of biological and non-biological research, this device aims to detect the fluorescent intensity of GFP and calculate the concentration of the chemicals (Cr (VI), beta-lactam, and tetracycline, respectively) detected based on it. We can then assess the quality of the sample detected.
The mechanism of this device is simple (figure). The exciting light coming from blue LED through 490nm narrowband filter, which only allow 490nm light to pass through. The filtered light then penetrates our test chip. If GFP exists on our test chip, it would transform the frequency of the excitation light into about 520nm, which is the only frequency of light that can pass through the 517nm narrowband filter. Then our sensor will be able to detect the intensity of the light and calculate the content of GFP, indicating the composition of the tested sample with our mathematical model.
The characteristic of our device is that it is really CHEAP. Although it costs less than 300RMB in total and can easily cooperate with our biological products.
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