Team:WLC-Milwaukee/Background
BACKGROUND
Introduction
The lack of clean water and regular water testing is an issue worldwide. These issues highlight the importance of researching a time and cost efficient water test. Lambda phage was our phage of choice to make our enzyme conjugate. We conjugated the J-protein from our phage with HRP, an enzyme with colorimetric and chemiluminescent properties. More information about our our phage and enzyme are included below.
Water Contamination and Testing (United States and Global)
Safe drinking water is a commodity that not all countries can afford. Many developing countries lack the resources needed to purify their source water and make them safe for the population. Living in the United States, many people take for granted the various efforts that are made to ensure that the public has safe water to drink. The CDC lists numerous contaminants which can infect our water, including microorganisms, disinfectants, and organic and inorganic compounds. Not only is water treatment an important step in protecting public health, but so is additional water testing in order to provide people with the safest drinking water possible. Drinking water in the United States comes from one of two sources: surface water or ground water. Surface water is found in lakes, rivers, and oceans, while ground water is found underground in crevices and spaces between rocks. Treated surface water is one of the most common types of water that Americans use. 1 Surface water is further purified with a disinfectant after treatment before it is available for the population to drink [1]. This method of water treatment applies to public water systems and is under the regulation of the Environmental Protection Agency (EPA), but what of water that comes from private wells? Distillation and filtration systems are normally put in place to filter out harmful impurities, but in many cases these precautions are not employed. In addition certain maintenance levels are required in order to keep the water supply safe to drink [2].
Water testing in public water systems involves testing for bacteria and parasites such as Escherichia coli and Cryptosporidium. Tests such as lactose fermentation methods can examine the number of coliform bacteria in a water sample. Often times, this is a good indicator of the sanitary quality of the water. Another way that water can be tested is by using a substrate to help detect contaminants. For instance, a substrate can specifically bind to products that particular bacteria may produce, thus detecting contamination [2]. While these methods may be conducted by water companies for public or private water sources, very few accurate test kits can easily be utilized at home. 3 Additonally, many of these water tests can take 24-48 hours for results to be obtained [3].
In most developing countries, water monitoring responsibilities belong to water suppliers or independent agencies. However, most developing countries do not meet the requirements for testing set by the World Health Organization due to lack of resources or time [2]. Because developing countries continue to use water sources that are not tested, dehydration, malnutrition, and waterborne diseases such as cholera and typhoid fever are common and are responsible for the death of 1 in 5 children under the age of 5 [1,4]. Low income developing countries are not the only ones that struggle with water contamination. The recreational water sources and popular beaches in Rio de Janeiro, Brazil, are contaminated by sewage which can lead to more serious water-related illnesses. Although the waters of Rio de Janeiro have been tested, these standard bacterial studies may no longer be sufficient and there may be a need for more specific pathogen testing [3]. Water contamination and safety is a huge problem in most countries. Clean and safe water is essential for human livelihood and happiness. While most countries test some of their water, most of the tests do not meet the guidelines set by the World Health Organization. It is important that we solve this issue of water safety and contamination by creating an economic and time efficient way to test water sources.
- [1] https://www.cdc.gov
- [2] https://www.epa.gov
- [3] https://watertestingkits.com/bacteria/
- [4] https://thewaterproject.org/why-water/health
- [5]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600167/
Lambda Phage
Lambda phage is a bacteriophage that can infect the well-known bacteria Escherichia coli. It was discovered in 1950 by microbiologist, Esther Lederberg, whose finding would lead to further investigation on gene regulation and recombination [1]. Lambda phage is composed of an icosahedral head and a flexible tail. Lambda phage infects E. coli by using its J protein on its tail to recognize the receptor integral outer membrane protein, LamB, which is found on the surface of E.coli. Research conducted by Karine Gibbs showed that LamB is not distributed uniformly throughout the outer membrane of E.coli, and in fact its distribution changes readily and quickly, illustrating the fluidity of the cell membrane [2]. Upon binding to the surface of E.coli, Lambda phage is able to inject its 48,502 bp of DNA into E. coli where it can either replicate or become a prophage [3]. This means that Lambda phage is capable of following the lytic pathway if it replicates, or the lysogenic pathway if it inserts its DNA into E. coli’s chromosome.
The discovery of Lambda phage has many important applications which have been utilized in past years and also continues to be researched on. For example, the water testing kit we are composing relies heavily on utilizing the binding specificity of lambda phage to E.coli in order to rapidly detect E. coli in water samples. Another use of Lambda phage is to use it for phage therapy, which simply means that Lambda phage is used to attack and kill E.coli [4]. More broadly, lambda phage is used as a model to investigate the mechanisms of biological processes, such as the specific binding of bacteriophage to bacteria. The importance of Lambda phage and its discovery has implications in both the health sciences and the environment, making it one of the most important bacteriophages in biotechnology.
- [1] http://www.whatisbiotechnology.org/index.php/people/summary/Lederberg_Esther
- [2] Gibbs, K. A., Isaac, D. D., Xu, J. , Hendrix, R. W., Silhavy, T. J. and Theriot, J. A. (2004), Complex spatial distribution and dynamics of an abundant Escherichia coli outer membrane protein, LamB. Molecular Microbiology, 53: 1771-1783. doi:10.1111/j.1365-2958.2004.04242.x
- [3] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509688/
- [4] https://brainmass.com/biology/cell-and-molecular-biology/lambda-phage-biotechnology-575620
Horse Radish Peroxidase
Horseradish peroxidase (HRP) is an enzyme used in molecular biology. It can be used in various biological methods, such as ELISA, Western Blotting, Southern Blotting, and EMSA [1]. HRP’s colorimetric and chemiluminescent properties make it a useful agent in many tests. HRP acts as a catalyst in redox reactions [3]. A luminol and H2O2 solution can release light for a very long time, but when HRP is added, it usually reaches it maximum light emission between 1 and 5 minutes [4]. This makes it a useful detection method. HRP can be used in more qualitative methods as well, since it induces changes in color when used with chromogenic assays.
Commonly used substrates for this type of detection are 3,3',5,5'-tetramethylbenzidine (TMB), 2,2' -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and diaminobenzidine (DAB) [3,4]. HRP when added to TMB, for example, yields a 2-electron oxidation product which yields a blue color. HRP is also up and coming in the medical field. When combined with other agents, it is heavily reactive toward tumor cells in humans [2]. It also serves as a functional tracer. HRP can be injected into the bloodstream and then identified at different places in the pathway using the DAB/H2O2 reaction [4]. This makes HRP very useful for immunocytochemistry and electron microscopy [4].
- [1] https://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=15552120
- [2] https://chem.libretexts.org/Textbook_Maps/Biological_Chemistry/Catalysts/Case_Studies/Horseradish_Peroxidase
- [3] http://www.bio-rad.com/featured/en/hrp-substrate.html
- [4] https://www.sciencedirect.com/topics/neuroscience/horseradish-peroxidase
- [5] http://www.jbc.org/content/257/7/3669.full.pdf
- [6] https://commons.wikimedia.org/w/index.php?curid=47268587 Manuel Almagro Rivas - Own work, CC BY-SA 4.0
