Team:Bielefeld-CeBiTec/Project/HeavyMetals
Heavy Metals
We detect several heavy metals with a single test strip.
Occurrence
Arsenic is found in nature in both organic and inorganic forms, typically as arsenite (AsIII) or arsenate (AsV). The average arsenic concentration in sea water is about 1-2 µg/L (Kaur et al. 2015). Inorganic arsenic is naturally present at high levels in the groundwater of a number of countries, including Argentina, Bangladesh, China, India, Mexico, and the USA (World Health Organization 2012). Arsenic contamination is most dramatic in Bangladesh, where over one million people suffer from arsenic poisoning. Strong local and seasonal fluctuations in arsenic concentrations make it necessary to test each well regularly (van der Meer 2003).
Health effects
Inorganic arsenic compounds are highly toxic. Acute effects of arsenic intake can range from gastrointestinal distress to death. Chronic exposure can result in skin lesions, vascular diseases and cancer. These chronic effects are referred to as arsenicosis, and there is no effective therapy for them. Due to its toxicity and frequency, arsenic ranks first on the Priority List of Hazardous Substances prepared by the US Environmental Protection Agency (EPA) and the Agency for Toxic Substances and Disease Registry (ATSDR). The World Health Organization recommends a limit of 10 µg/L in drinking water, but some countries have adopted a national standard of 50 µg/L (World Health Organization 2012; Chen, Rosen 2014).
Detection
Arsenic can be accurately detected by means of techniques such as atomic absorption spectroscopy (AAS), atomic fluorescence spectrometry or high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, they are expensive and not suitable for field testing (Chen, Rosen 2014). Chemical test kits are available, which mostly rely on the Gutzeit method. This method is based on the generation of arsine gas from a sample solution. Arsine then reacts with a mercuric bromide impregnated test strip, which results in a color change (Kearns 2010). The accuracy and reliability of this method has been called into question (Rahman et al. 2002). The need for an inexpensive and realible detecion method has led to the development of various arsenic biosensors. Among them are both whole-cell-based and cell-free biosensors. For a recent review, refer to Kaur et al. 2015.
Our arsenic biosensor
We choose to work with the chromosomal arsenic operon of E. coli, which was used by the team from Edinburgh in 2006. This operon encodes an efflux pump which confers resistance against arsenic. The expression is controlled by the repressor ArsR, which negatively autoregulates its own expression. AsIII can bind to three cysteine residues in ArsR. The resulting conformational change deactivates the repressor (Chen, Rosen 2014).
References
Chen, Jian; Rosen, Barry P. (2014): Biosensors for inorganic and organic arsenicals. In Biosensors 4 (4), pp. 494–512. DOI: 10.3390/bios4040494.
Kaur, Hardeep; Kumar, Rabindra; Babu, J. Nagendra; Mittal, Sunil (2015): Advances in arsenic biosensor development--a comprehensive review. In Biosensors & bioelectronics 63, pp. 533–545. DOI: 10.1016/j.bios.2014.08.003.
Kearns, James Kalman (2010): Field Portable Methods for the Determination of Arsenic in Environmental Samples. Dissertation.
Rahman, Mohammad Mahmudur; Mukherjee, Debapriyo; Sengupta, Mrinal Kumar; Chowdhury, Uttam Kumar; Lodh, Dilip; Chanda, Chitta Ranjan et al. (2002): Effectiveness and Reliability of Arsenic Field Testing Kits: Are the Million Dollar Screening Projects Effective or Not? In Environ. Sci. Technol. 36 (24), pp. 5385–5394. DOI: 10.1021/es020591o.
van der Meer, Jan Roelof (2003): EAWAG news 56e: Bacterial Biosensors to Measure Arsenic in Potable Water.
World Health Organization (2012): Arsenic fact sheet. Available online at http://www.who.int/mediacentre/factsheets/fs372/en/, checked on 8/12/2015.
Occurrence
Chromium is an essential part of the earth´s crust.It is the sixth most abundant one and used inmetallurgical, chemical and refractory form. The three most important oxidative forms of chromium are the elemental metal (Cr), the trivalent (CrIII) and the hexavalent(CrVI) (Mitchell D. Cohen et al.).
Health effects
While the trivalent form is an essential dietary mineral and themost common natural form, we are interested in the hexavalent form because of its potential toxicity and carcinogenetic effects. Most of it is produced trough industrial uses(Paustenbach et al. 2003). Chromium intoxication can result in damage to the nervous system, fatigue and mental instability (Singh et al. 2011). It´s potential cancerogenity results out of chromium VI being able to enter the cells while it is not possible for chromium III compounds. Chromium VI in the cells is reduced to chromium III and can´t leave the cells anymore (Mitchell D. Cohen et al.). Because of its toxicity the World Health Organization (WHO) recommends a limit of 50 µg/l chromium in drinking water. In contrast to this guideline concentrations as high as 120µg/l chromium were detected in drinking water (Guidelines for drinking-water quality 2011) .
Detection
Chromium in drinking water is detected trough atomic absorption spectroscopy (AAS) or Ion chromatography with post column derivatization and UV visible spectroscopic detection (U.S. EPA, OW, OGWDW, SRMD, Technical Support Center). Moreover chromium detection at home can be detected by a basic titrimetric method using a iodide reaction for measurement (GIORGIA).
Our chromium biosensor
We work with the chromate inducible operon of Ochrobactrumtritici5bvl1which enables a resistance for chromium VI and superoxide which was introduced to iGEM by BIT 2013 team. The parts which are of interest to us are the Chromium dependent Repressor ChrB and its associated Promoter (Branco et al. 2008) The output of our sensor system works through fluorescence.
References
Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016. cavillona (2005): Nickel in Drinking-water, checked on 9/9/2015. EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015. Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015. Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x. kreusche: Trinkwasser-Installation 27.6.07 Endfassung.qxd, checked on 9/10/2015. US: Technical Factsheet on: Nickel, checked on 9/9/2015.
Occurrence
Copper is an essential trace element for humans’ animals and plants. In the human body you have concentrations of 1.4 to 2.1 mg/kg body mass. Copper is ingested through the gut and transferred to most tissues trough the liver. It is used present in coatings and alloys and used to make pipes, valves and fittings. Moreover coppersulfate pentahydrate is used in algae control by adding it to surface water. Therefore copper concentrations in drinking water vary widely (Guidelines for Drinking-water Quality, Fourth Edition)
Health effects
Copper is an essential for human health, but in to high doses it can cause anemia, kidney and liver damage as well as stomach and intestinal irritation and immunotoxicity(ATSDR). Some people are at greater risks off negative health effects caused by copper overexposure. A with copper associated disease is Wilsons disease which results in a miss function, so that copper can´t be excreted by the liver into bile.If it´s not treated it can result in brain and liver damage.(US EPA ORD NCEA Integrated Risk Information System (IRIS) 2014)Some studies associate high copper levels with aging diseases as Atherosclerosis andAlzheimer’s Disease(Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library)
Detection
The most important analytical methods for the detection of copper in water areinductively coupled plasma mass spectrometry (ICP-MS) which has the lowest detection limit (0.02 μg/litre) and Atomicabsorption spectrometry (AAS) with flame detection, which has the highest (20 μg/litre) as well as graphite furnace atomicabsorption spectroscopy, inductively coupled plasma atomic emission spectroscopy,and stabilized temperatureplatform graphite furnace atomic absorption(cavillona 2004).
Our copper Biosensor
The operon we used for our biosensor is native to E.coli K12. The parts we used are CopA promoter (CopAP) and it´s regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-ype ATPase pump(Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to periplasm. This is possible trough Copper induced activation of the operons transcription via CueR.CueR-Cu+ is the DNA-binding transcriptional dual regulator which activates transcription(Yamamoto, Ishihama 2005) To sum it up CueR regulon plays an important role in aerobic copper tolerance in E.coli(Grass, Rensing 2001).We combined CueR under the control of a constitutive promoter with CopAP a 5´ untranslated region for higher translation levels and sfGFP for measuring output signals
References
ATSDR: TOXICOLOGICAL PROFILE FOR COPPER, checked on 8/27/2015. cavillona (2004): Copper in Drinking-water. Background document for development of WHO Guidelines for Drinking-water Quality, checked on 9/9/2015. Copper excess, zinc deficiency, and cognition loss in Alzheimer's disease - Brewer - 2012 - BioFactors - Wiley Online Library. Available online at http://onlinelibrary.wiley.com/doi/10.1002/biof.1005/abstract, checked on 8/28/2015. Grass, Gregor; Rensing, Christopher (2001): Genes Involved in Copper Homeostasis in Escherichia coli, checked on 8/26/2015. Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015. Outten, F. W.; Outten, C. E.; Hale, J.; O'Halloran, T. V. (2000): Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. In The Journal of biological chemistry 275 (40), pp. 31024–31029. DOI: 10.1074/jbc.M006508200. US EPA ORD NCEA Integrated Risk Information System (IRIS) (2014): Copper (CASRN 7440-50-8) | IRIS | US EPA. Available online at http://www.epa.gov/iris/subst/0368.htm, updated on 10/31/2014, checked on 9/2/2015. Yamamoto, Kaneyoshi; Ishihama, Akira (2005): Transcriptional response of Escherichia coli to external copper. In Molecular microbiology 56 (1), pp. 215–227. DOI: 10.1111/j.1365-2958.2005.04532.x.
Occurrence
Lead is a heavy metal. Its widespread occurrence, relatively simple extraction and combination of desirable properties have made it useful to humans. But the frequency of occurrence is not as high as that of other metals. Nonetheless lead plays a major role in the industryand is one of the most used metals. For example, lead is important for the manufacture of batteries, which are used in vehicles. Lead is found in different parts of the environment, so it is in the air, the soil and in the water. Lead and lead compounds are used in a high variety of products, which are found in pipes and plumbing materials, solders, gasoline, batteries, ammunition and cosmetics, just as product that is found in and around our homes. Due to the fact that lead is also occurring in water, if there are obstruct pipes that consist of lead or that has a part of lead, respectively.When lead is used in household plumbing, this allows water to be easily contaminated with the metal.Long time absorption could results in adverse health effects.
Health Effects
Lead has no biological role in the body, but it is a highly poisonous metal. The ingestion of lead could affect almost every organ and system in the body (Heavy Metals Testing ByUsp. Caspharma.com.). The main target for lead toxicity is the nervous system.It could have acute or chronic health effects. The acute health effects are occurring immediately after contact with lead. So it can irritate the eyes or can lead to headache, irritability, disturbed sleep, and mood and personality changes. Exposure to higher lead content over a long-term could cause serious damage to the brain and to the kidneys. The damage can finally cause death (Golub, M. S., 2005). The poisoning is mostly resulting of ingestion of water or food, which is contaminated with lead or lead compounds (Ferner D. J., 2001).It is taken up fast in the bloodstream and spread in the body(Bergeson, 2008). The World Health Organization recommends a limit of 10 µg/L in drinking water, concentrations in drinking water are generally below 5 μg/l. But there are much higher concentrations that have been measured if lead fittings are existing (WHO: Guidelines for Drinking-water Quality,fourth edition).
Detection
Due to the effects on health the detection of lead in drinking water is of importance in all parts of the world. So, a simple system for fast review of the water quality is a worthwhile aim. The method for detection is currently on the principle of flame atomic absorption spectrometry (FAAS) (Abdallah, A. T. and Moustafa, M. A.,2002). This detection method is impossible in developing countries, for example, thus there are hardly quality standards.
Our lead biosensor
For our biosensor, we use the chromosomal lead operon of Cupriavidusmetallidurans(Ralstoniametallidurans). The promoter that we use is PbrA. This part of the operon is regulated by the repressor pbrR. The PbrR protein is mediated Pb2+-inducible transcription. PbrR belongs to the MerR family, which are metal-sensing regulatoryproteins (Borremanset al., 2001). Our sensor system comprised PbrR, which is under the control of aconstitutive Promoter and PbrA and a 5’ untranslated region, which controls the transcription of a sfGFP. The sfGFP protein is the measuring output signal.
References
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Occurrence
The amount of natural occurring nickel is quite low even if it is anelement of the earth’s crust. Therefore small amounts of it are found in food water soil and air. Nickel-concentration in drinking water is normally less than 0.02 mg/l, although troughreleases from taps and fittings the nickel may contribute to concentrations up to 1 mg/l. There may be higher concentrations in drinking-water in special cases of release from natural or industrial nickel deposits in theground and therefore a higher guideline value of 0.07 mg/l (70 μg/l)(Guidelines for Drinking-water Quality, Fourth Edition)
Health effects
Evan if nickel is essential for mammals and part of human nutrition it may cause dermatitis as well as itching of fingers, hands and forearms in some people who had long term skin contact. The main source of nickel exposure is food or water but most people have contact to nickel trough everyday products as jewelry or stainless steel dishware or trough smoking tobacco(US; EPA et al. 2013).In Germany most drinking water pollutions by nickel happen in the last meters of the plumbing system. Wrong tapware is the main source of nickel contamination in drinking water (kreusche)
Detection
The two most commonly used analytical methods for nickel in water are atomicabsorption spectrometry and inductively coupled plasma atomic emissionspectrometry. Inductively coupled plasma atomic emissionspectroscopy is used for the determination of nickel etection limit ofabout 10 μg/litre (ISO, 1996). Flame atomic absorption spectrometry is suitable in the range of 0.5–100 μg/litre (ISO, 1986). A limit of detection of 0.1 μg/ can be achieved using inductively coupled plasma mass spectrometry. Alternatively, electrothermal atomic absorption spectrometry can beused. (cavillona 2005)
Our nickel biosensor
For our nickel sensor system we used the rcn-operon from E. coli which codes for a nickel- and cobalt-efflux system, which is highly sensitive to nickel . If Ni(II)-ions bind to the repressor RcnR, it cannot attach to DNA and RcnA the nickel responsive promoter is activated. In the absence of nickel or cobalt, RcnR is bound to RcnR operator and blocks RcnA transcription. (EPA et al. 2013; Blaha et al. 2011; Iwig et al. 2006) Our output signal works trough fluorescence.
References
Blaha, Didier; Arous, Safia; Blériot, Camille; Dorel, Corinne; Mandrand-Berthelot, Marie-Andrée; Rodrigue, Agnès (2011): The Escherichia coli metallo-regulator RcnR represses rcnA and rcnR transcription through binding on a shared operator site: Insights into regulatory specificity towards nickel and cobalt. In Biochimie 93 (3), pp. 434–439. DOI: 10.1016/j.biochi.2010.10.016. cavillona (2005): Nickel in Drinking-water, checked on 9/9/2015. EPA, U. S.; OAR; Office of Air Quality Planning and Standards (2013): Nickle Compounds | Technology Transfer Network Air Toxics Web site | US EPA. Available online at http://www.epa.gov/airtoxics/hlthef/nickel.html, updated on 10/18/2013, checked on 9/10/2015. Guidelines for Drinking-water Quality, Fourth Edition, checked on 9/9/2015. Iwig, Jeffrey S.; Rowe, Jessica L.; Chivers, Peter T. (2006): Nickel homeostasis in Escherichia coli - the rcnR-rcnA efflux pathway and its linkage to NikR function. In Molecular microbiology 62 (1), pp. 252–262. DOI: 10.1111/j.1365-2958.2006.05369.x. kreusche: Trinkwasser-Installation 27.6.07 Endfassung.qxd, checked on 9/10/2015. US: Technical Factsheet on: Nickel, checked on 9/9/2015.
