Nowadays, the detection of substances becomes an important technology that has a vast variety of applications, including the monitor of water quality, food security, which are all closely related to our daily life. Consequently, there's also an increasing demand in the market for more affordable and efficient detectors.
Nowadays, the detection of substances becomes an important technology that has a vast variety of applications, including the monitor of water quality, food security, which are all closely related to our daily life. Consequently, there's also an increasing demand in the market for more affordable and efficient detectors.
Existing chemical method used to detect metal or non-metal elements possess a common character of high cost and high complexity. For instance, inductively coupled plasma-optical emission spectroscopy (ICP-OES) is an analytical technique used to identify and analyze a sample for its elemental content. However, ICP-OES requires costly instrumentation for plasma generation, sample aerosolizing, and signal analysis, total cost ranging from $60,000 to $100,000 (A., 2017). The expense may be prohibitive to many laboratories with inadequate funding. (M.,2021)
Elemental analysis based on physical phenomena or processes includes X-ray emission spectroscopy (XES), Mass spectrometry (MS) is an analytical laboratory technique used to identify the components of a sample by their mass and electrical charge. Using MS to detect chemical elements, however, involve the use of a specialized instrument: mass spectrometer, whose price ranging from $10,000 to nearly $100,000 (Excedr, 2022). Specialized training is also demanded to operate such sophisticated apparatus, bringing great inconvenience for unprofessional researchers or users. (T.& V., 2000)
A number of biological detectors, such as GFP-based biosensors, have been developed in the past few years. Green fluorescent protein (GFP), for example, is a powerful reporter protein that allow labeling and identifying specific cells or elements. However,the high price of spectrophotometers used to detect fluorescent proteins and the requirements for specialized operations still made them commercially unavailable. Admittedly, current common detection methods, as shown above, may possess high specificity and sensitivity, but they remains not only costly and time-consuming, but also requires high professionalism to operate.Therefore, THINKER_CHINA aimed to develop a detection method cheaper and more efficient than the existing ones.
We hope to design a biological detector that detects and reports tested ions in aqueous solution by utilizing the tolerance mechanism of bacteria to various chemical elements. Since bacteria's metabolic precursors and regulatory factors have specific reactions to different ions, using these reactions, we can qualitatively detect and report the intensity of certain factors need to be tested. In this experiment, we selected arabinoses and copper ions as tested ions and synthesized operons corresponding to these ions.
Our intention is to make a low-budget, efficient and user-friendly chemical detector.
As the flowchart above shows, it consists of 3 promoters, 2 RBS, 2 Terminator with 1 gene and a beta-galactosidase.
Promoter is the target sequence of RNA polymerase and some transcription factors, regulates and controls transcription initiation. While we used T7 promoter as a strong promoter from T7 phage.
Arabinose is a five-carbon sugar that is found widely in nature and can serve as a sole carbon source in many bacteria. Moreover, Thinker_China has also standardized and characterized its regulatory circuits.
Cu-sensitive promoter: This nucleotide sequence is believed to bind to phosphorylated CusR transcription factors in Escherichia coli. In the presence of high extracellular concentrations of copper ions, CusR proteins are phosphorylated by CusS transmembrane proteins. After phosphorylation, CusR and the DNA sequences interact with each other and activate CusA, CusB, CusC, and proteins encoding proteins of the copper metabolic system Transcription of CusF gene.
We chosed BBa_B0034 as RBS, which is a RNA sequence found in mRNA to which ribosomes can bind and initiate translation.
Terminator is the DNA sequence that gives the transcription termination signal to the RNA polymerase, located downstream of the poly (A) site, The terminator we used is BBa_B0015, a double terminator consisting of BBa_B0010 and BBa_B0012.
The SRRZ cleavage gene we used is a linked gene is composed of perforin gene S, bacteriophage lysozyme (transglycosidase) gene R and gene RZ.
The product of the R gene is a water-soluble transglycosylase, a peptidoglycan that breaks down cell walls.
The product of the RZ gene is an endopeptidase that cleaves between the oligosaccharides of the peptidoglycan and cross-links between the peptidoglycan and the outer membrane of the cell wall.
The function of the S gene product is to change the permeability of the plasma membrane and form a porous structure on the plasma membrane so that the enzymes produced by R and RZ genes can cross the plasma membrane and reach the cell wall, thus acting on the cell wall, so that the cell wall broken, the release of intracellular substances.
The function of both the R and RZ gene product is to degrade the cell wall.
β-Galactosidase is a glycoside hydrolase enzyme that catalyzes the following process: Hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
Our system is made up of 2 main parts: chromogenic system and bacteria lysis system.
Phage has its gene sequence contained a SRRZ-lysing gene, whose expression effect is to dissolve bacterial cell wall, so that viruses can be injected into bacteria. Applying this reaction into our experiment, the promotors added will cause rapid expression of the SRRz lysis gene as well as the dissolution of the bacterial cell wall.
Take copper iron promotor as an example. Before our bacterial strain works, the T7 constitutive promoter starts transcribing and translating the Beytagh Galactosidases and stores the enzyme in the cell. When testing is about to process, the tested sampleis are added to the strain container. The Cu ion promoter initiates the lysis of the gene, releasing the beta-galactosidases, which then interacts with the X-gal Galactosidases to produce the blue substance that indicates the presense of copper.
Due to bacteria lysis, its bacterial endogenous ß-galactosidase inside is released into the environmental solution, which catalyzes colorization reaction of colorless substrate X-Gal or oNPG. , the grey value of colorization reaction shows obvious regularity.
The scale of the color generated by the chromic reaction is positively correlated with the cell lysis rate, which is positively correlated with the expression intensity of SRRz gene, which is induced by the operon, and the operon is positively correlated with the signal intensity of tested ions. Therefore, different tested ion intensities lead to different grayscale values of the final color.
Simple procedure and instrument and portable(all)
Our detector system is designed to be in a small volume, consequently, it can be placed without taking up much space, which in turn contribute to its low transport cost. In addition, the apparatus used for detection, including ( ), are mostly affordable scientific instruments.
Unlike the vast majority of instruments that possess complex operation process, our biodetector can be applied in a more time-saving way with only three procedures. After that, we can apparently recognize how is the density of various objects under inspection through their color variance in a few seconds.
Furthermore, throughout our detection process, little professional knowledge is required to operate the instruments, which enables non-professionals to carry out the whole testing procedure following several simple instructions. This largely saves the potential spendings on training for non-scientist or recruitment of professionals.
Our project aims to encourage the applications of this innovative technology in more areas: From the medical aspect, for example, this technology can be used to detect uric acid, white blood cells and other human molecular indicators, so as to realize the examination of health status or even basic diagnosis of diseases;from the medical aspect, this technology can be used to detect uric acid, white blood cells and other human molecular indicators, so as to realize the detection of health status and basic diagnosis of diseases; Moreover, in terms of environmental protection, our detection system can identify and monitor pollutants in water and soil, enable people to make corresponding plans to help alleviate the prevailing pollution problem in cetain regions. Beyond that, our biodetector have numerous other uses, such as the detection or analysis of heavy metals, nutrients, plasma concentration, etc.
Our elementary analysis system is not only meant to serve the minority of professionals, but also anyone who wishes to apply our project into various domains, and translate the seemingly obscure and unreachable technologies into actual social and environmental benefits.
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