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 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 asfGFP for measuring output signals
Publication bibliography
Publication bibliography 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.
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