Difference between revisions of "Team:Utrecht/Description"

 
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<h1> Project Description </h1>
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<p>Humans have been polluting water with natural products for hundreds of years. However, during the last decennia the amount of unnatural chemicals that are being produced, used and carelessly disposed, rapidly increases. The disastrous effects of water pollution by emerging contaminants on animals, humans and environment becomes more and more evident.</p>  
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<p>Emerging contaminants are pollutants with a rising concern, for example ecological systems and human health.
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Around a 140.000 kilograms of medical waste a year is present in dutch waters. This leads to huge consequences in ecological systems and human health (Joost van Kasteren, 2016; Nieuwenhuis, 2016). One of the biggest source of the pollution are hospitals (Belfroid et al., 1999). “The possible effects of pharmaceuticals include behavioral changes, tissue damage and effects on reproduction of water organisms, as a result of which the ecosystem as a whole may be disrupted.”- RIVM (Moermond, Smit, van Leerdam, van der Aa, N. G. F. M., & montforts, M. H. M. M., 2016).
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The first step in analysing and mitigating the effects is to detect these contaminants in water and locate the source of the contamination.</p>
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<p>Currently, a set of biosensors are used. These however are not specific at all. Only stress is detected, after which an alarm will ring. Examples of these biosensors are algae and daphnia. As another disadvantage, the biosensor might get used the pollution, resulting in needing a higher concentration of contaminants before performing stress (Epema, 2018). </p>
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<p>Another example is the microtox-test. It is based on a bioluminescent bacteria: Photobacterium phosphoreum. The bacterium is continuously luminescent, trough metabolizing organic compounds. When stressed by pollutions/contimaniants the bacterium stops metabolising light. If the signal gets below 50%, pollution is confirmed. These pollution however, is quite broad and does not give an indication of the kind of contaminant or any idea about the concentration (ALS global, 2018; Johnson, 2005).</p>
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<p>These methods are used by rijkswaterstaat, a company that controls water quality in surface water. For these controls, a limit in concentration is set. If this limit is exceeded, purification of the water needs the find place (Epema, 2018).</p>
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<p>But before surface water becomes surface water, it is cleaned by sewage treatment. However, sewage treatment only focuses on cleaning organic compounds, neglecting the pharmaceuticals. An essay indicating which groups of compounds that get in the treatment and out is valuable information indicating the efficiency of the treatment. The ideal biosensor would be one that  can detect specific groups of compounds at once with a direct indication of concentration. With an indication of the toxic compound the search in the mass spectrometer is much easier. This is where our biosensor comes in.</p>
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<p id="part2">We will be using the chemotaxis pathway of the bacteria Escherichia coli. The chemotaxis pathway allows the bacterium to swim towards substances it needs to survive and is mediated by a highly conserved, specific, and well-studied pathway. It consists of a two-component system, a tumbling state, in which the bacteria awaits new stimuli, and a running state, in which it responds to a detected stimulus. When the receptor has no attractant bound, the chemotaxis pathway is constitutively active. In this case the Tar receptor activates the kinase CheA. CheA subsequently transfers its phosphoryl group to the response regulator CheY. Phosphorylated CheY (CheY-P) translocates to the flagellar motor, where it interacts with motor proteins FliM and FliN. This causes the flagellar motor to change its rotational direction, causing the bacterium to tumble. CheY-P is dephosphorylated by CheZ. Upon binding of an attractant, the Tar receptor becomes inactivated and the equilibrium of the pathway is set to low activity, causing the bacterium to switch to a running state.</p>
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<p>We will perform multiple adjustments to make our biosensor as good as possible.</p>
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<center><img src="https://static.igem.org/mediawiki/2018/5/53/T--Utrecht--2018--Chemotaxis_Pathway--Main002.png" alt="Chemotaxis_Pathway.png" width = 50% height = auto></center>
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<p> As a first step a light signal will be coupled to the binding of a substance with the receptor. A combination of LuxAB and eYFP is used to make BRET (Bioluminescence Resonance Energy Transfer).  In close proximity, luxAB will activate eYFP. EYFP activation results in a detectable light. LuxAB and eYFP proteins are coupled to cq. CheZ and CheY. When no substance is bound, CheY and CheZ bind, causing a light signal to appear.  If a substance binds, the light signal will disappear. The decrease in light signal is a method to detect whether a compound has bind or not.</p>
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<p>Secondly we will customize the sensory domain while the rest of the pathway remains intact. As new sensory domain, the adrenaline receptor is used. With this receptor we hope to detect adrenaline and adrenaline-like substances. This concept provides a more broad range of detection with one receptor. Later we hope to change the sensory domain to multiple receptors, allowing to detect more groups of contaminants in water. The ligand binding domains of the cytokinin receptor PcrK, and the epinephrine receptor QseC are similar to the intracellular domain of the E. coli Tar receptor. We opt to use three different fusion points for both receptors based on previously published successful recombinant chemotaxis receptors. We will modify these sensors so they can measure different concentrations of ligand by modifying the methyl accepting residues of the Tar methylation helixes, and expressing different levels of recombinant receptor.</p>
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<p>Finally the sensory domain of the biosensor is methylated on 4 sites, resulting in a change in affinity of the receptor. This allows us to measure different concentrations. The methylations will be accomplished with SDM (site directed mutagenesis).</p>
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<p>In conclusion, our biosensor is a cost-effective and durable method to measure emerging contaminants in drinking or surface water. This can be used to detect which contaminants are present and can function as a control step to check if the purification of  water succeeded. It will also provide an indication of the concentration level.</p>
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<h3>Project Description</h3>
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<li>ALS global. (2018). EnviroMail™ 120 - microtox toxicity test. Retrieved from <a href="https://www.alsglobal.com/myals/news/2018/02/enviromail-120-microtox-toxicity-test">https://www.alsglobal.com/myals/news/2018/02/enviromail-120-microtox-toxicity-test </a></li>
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<p>Humans have been polluting water with natural products for hundreds of years. However, during the last decennia the amount of unnatural chemicals that are being produced, used and carelessly disposed of, has been increasing rapidly. A substantial part of these chemicals introduced in our water supplies are derived from pharmaceuticals.</p>
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<li>Belfroid, A. C., Van der Horst, A., Vethaak, A. D., Schäfer, A. J., Rijs, G. B. J., Wegener, J., & Cofino, W. P. (1999). Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the netherlands. Science of the Total Environment, 225(1), 101-108. 10.1016/S0048-9697(98)00336-2 Retrieved from <a href="http://www.sciencedirect.com/science/article/pii/S0048969798003362">http://www.sciencedirect.com/science/article/pii/S0048969798003362</a></li>
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<li>Epema, O. (2018). Analytical chemist, head of laboratory of inorganic analysis at rijkswaterstaat</li>
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<p>We focussed on the one pharmaceutical contaminant that is associated with current day lifestyle, Selective Serotonin Reuptake Inhibitors (SSRI), commonly known as antidepressants. After leaving the human body, the remainder of the medicine ends up in the sewage system and is not broken down readily by the sewage treatment plants, resulting in an increasing concentration in surface waters. Even in low concentrations, antidepressant waste products pose a threat to ecological systems, for example because they are still biologically active in vertebrates, changing their behaviour and fertility. Current techniques for the detection of pharmaceutical waste products are either expensive or inaccurate. An improved system is therefore needed.</p>
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<li>Johnson, B. T. (2005). Microtox® acute toxicity test. In C. Blaise, & J. Férard (Eds.), Small-scale freshwater toxicity investigations: Volume 1 - toxicity test methods (pp. 69-105) Springer Netherlands. Retrieved from //ww.springer.com/la/book/9781402031199</li>
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<li>Joost van Kasteren. (2016). 140.000 kilo medicijnresten per jaar, in ons water. hoe krijgen we die eruit? Retrieved from <a href ="https://www.nrc.nl/nieuws/2016/11/26/met-ozon-bubbel-je-slaappil-uit-het-water-5523164-a1533689">https://www.nrc.nl/nieuws/2016/11/26/met-ozon-bubbel-je-slaappil-uit-het-water-5523164-a1533689</a></li>
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<p>Consultation of experts and stakeholders provided us with five core requirements:</p>
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<li>Moermond, C. T. A., Smit, C. E., van Leerdam, R. C., van der Aa, N. G. F. M., & montforts, M. H. M. M. (2016). Geneesmiddelen en waterkwaliteit. ().</li>
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<li>Nieuwenhuis, M. (2016). RIVM: Medicijnen maken het water ziek. Retrieved from <a href="https://www.ad.nl/binnenland/rivm-medicijnen-maken-het-water-ziek~a89fa38e"> https://www.ad.nl/binnenland/rivm-medicijnen-maken-het-water-ziek~a89fa38e</a></li>
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<li>Possibility for detecting diverse compounds</li>
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<li>Accurate detection at different concentrations</li>
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<li>Clear and easily measurable detection signal</li>
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<li>Rapid signaling</li>
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<li>Low-cost system</li>
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<p>These requirements were met by developing DeTaXion, a biodetection system that exploits the fast and highly customizable chemotaxis pathway of <i>Escherichia coli</i>, a bacterium that can be produced at low cost. This pathway was adapted using a three step approach to accomplish the desired biosensor. First, exchanging the ligand binding domain of the <i>E. coli</i> chemotaxis receptor Tar will allow the bacteria to recognize diverse pharmaceutical compounds. We have already developed and implemented a validation assay to assess the functionality of the receptor. Next, methylation sites can be customized to extend the detection range of the DeTaXion biosensor which allows accurate pollutant detection at different concentration ranges. We have mathematically modelled the effect of changes in the methylation sites to assess the effect on the detection range. Finally, a bioluminescence resonance energy transfer (BRET) pair was introduced to generate an easily detectable and quantifiable signal.</p>
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<p>The mutated <i>E. coli</i> was designed to be integrated into an easily usable hand-held device. Our design includes various features to solve safety, regulatory, and technical issues. Ultimately, the product will enable low-cost, rapid, and accurate detection of pharmaceutical waste products for diverse ligands and at a range of concentrations. It can be used as a stand-alone product to continuously monitor a water supply, or to complement traditional chemical analyses, thereby improving the detection of pharmaceutical waste products in aquatic systems and contribute to a cleaner environment.</p>
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Latest revision as of 23:06, 17 October 2018

Description Safety Model BRETAssay EndProduct Biobricks Receptor Methylation

Project Description

Humans have been polluting water with natural products for hundreds of years. However, during the last decennia the amount of unnatural chemicals that are being produced, used and carelessly disposed of, has been increasing rapidly. A substantial part of these chemicals introduced in our water supplies are derived from pharmaceuticals.

We focussed on the one pharmaceutical contaminant that is associated with current day lifestyle, Selective Serotonin Reuptake Inhibitors (SSRI), commonly known as antidepressants. After leaving the human body, the remainder of the medicine ends up in the sewage system and is not broken down readily by the sewage treatment plants, resulting in an increasing concentration in surface waters. Even in low concentrations, antidepressant waste products pose a threat to ecological systems, for example because they are still biologically active in vertebrates, changing their behaviour and fertility. Current techniques for the detection of pharmaceutical waste products are either expensive or inaccurate. An improved system is therefore needed.

Consultation of experts and stakeholders provided us with five core requirements:

  1. Possibility for detecting diverse compounds
  2. Accurate detection at different concentrations
  3. Clear and easily measurable detection signal
  4. Rapid signaling
  5. Low-cost system

These requirements were met by developing DeTaXion, a biodetection system that exploits the fast and highly customizable chemotaxis pathway of Escherichia coli, a bacterium that can be produced at low cost. This pathway was adapted using a three step approach to accomplish the desired biosensor. First, exchanging the ligand binding domain of the E. coli chemotaxis receptor Tar will allow the bacteria to recognize diverse pharmaceutical compounds. We have already developed and implemented a validation assay to assess the functionality of the receptor. Next, methylation sites can be customized to extend the detection range of the DeTaXion biosensor which allows accurate pollutant detection at different concentration ranges. We have mathematically modelled the effect of changes in the methylation sites to assess the effect on the detection range. Finally, a bioluminescence resonance energy transfer (BRET) pair was introduced to generate an easily detectable and quantifiable signal.

The mutated E. coli was designed to be integrated into an easily usable hand-held device. Our design includes various features to solve safety, regulatory, and technical issues. Ultimately, the product will enable low-cost, rapid, and accurate detection of pharmaceutical waste products for diverse ligands and at a range of concentrations. It can be used as a stand-alone product to continuously monitor a water supply, or to complement traditional chemical analyses, thereby improving the detection of pharmaceutical waste products in aquatic systems and contribute to a cleaner environment.

Team
Project Design
Integrated Human Practices