Team:Bielefeld-CeBiTec/Project/HeavyMetals
Heavy Metals
We detect several heavy metals with a single test strip.
Heavy metals have been part in a lot of iGEM projects over the last years, so why work with them again?
Heavy metals Heavy metals are part of earth’s crust and therefore natural occurring in our environment. (Heavy Metals - Lenntech) In low doses some of them as copper or nickel are even essential trace elements for animals and humans (Rashmi Verma and Pratima Dwivedi 2013). A major problem is their bioaccumulation which leads to toxicity and long term effects which include fatal diseases as cancer (Martin et al. 2009) Parkinson or Alzheimer’s disease (Gaggelli et al. 2006).
Our biosensors We decided to work with already existing well characterized sensors as well as working with established but not well characterized concepts of other teams and moreover created new sensor systems. Therefore we established a basic construction plan for our sensor systems which is based on a promoter with a specific operator region in front of a super folder GFP (sfGFP) that was used for detection trough fluorescence analysis. In addition we used fitting activators or repressors for our inducible promoters under the control of BBa_K608002 which consists of a constitutive promoter with a strong ribosomal binding site (RBS). We combined these into a device consisting of constitutive promoter and RBS reverse and the promoter and operator region in front of the sfGFP. So we have repressor or activator constitutively express but reverse, to minimize background transcription of the inducible system in front of our heavy metal promoter operator system. In addition, these devices are optimized for the usage in a cell free protein synthesis (CFPS). This is the basis for the development of cell free biosensors on a test strip, that can be used to detect several heavy metals at one in the open field.
Detected heavy metals The heavy metal sensors we choose for detection are specific to arsenic, copper, chromium, lead, mercury and nickel. Which concentrations in drinking water are regulated by the WHO, because of their immediate and longtime health effects.
Our motivation We aim to make a use of well characterized sensors as well as concepts and new ideas. All this sensor systems shell work on the same principle, so that we can use them to create a modular easy to handle paper based cell free test strip for detection of more substances, heavy metals in this case, in parallel.
Click on the test strip for more information about the heavy metals and how they can be detected:
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 reliable detection 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 in metallurgical, 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 the most common natural form, we are interested in the hexavalent form because of its potential toxicity and carcinogenic 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 is the result of chromium VI being , able to enter cells , while this is not possible for chromium III compounds. Inside of the cells, chromium IV is reduced to chromium III and can not 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 in the USA (Guidelines for drinking-water quality 2011, WHO 2003).
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 Ochrobactrum triti ci5bvl1 which enables a resistance for chromium VI and superoxide . For the Sensor the Cr(VI) dependent repressor chrB and its associated promoter are needed (Branco et al. 2008). They were introduced by team BIT 2013 (BBa_K1058007 and BBa_K1058008). The output of our sensor system works through fluorescence generated with sfGFP. Our repressor is codon-optimised for use in E.coli.
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. The human body contains 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 in coatings 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 and range from 0,005 mg/L to 30 mg/L. After the Guidlines for drinking water the maximal concentration of copper is 2mg/L (Guidelines for Drinking-water Quality, Fourth Edition ).
Health effects
Copper is 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). A with copper associated disease is Wilsons disease which results in a miss function, so that copper can not be excreted by the liver into bile. If it is 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 and Alzheimer’s Disease (Brewer 2012).
Detection
The most important analytical methods for the detection of copper in water are inductively coupled plasma mass spectrometry (ICP-MS) which has the lowest detection limit (0.02 μg/L). Other often used methods for detection are Atomicabsorption spectrometry (AAS) with flame detection, which has the highest (20 μg/L) as well as graphite furnace atomicabsorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, and stabilized temperature platform graphite furnace atomic absorption (cavillona 2004).
Our copper Biosensor
We used the native operator of cooper homeostasis from E.coli K12. >This includes the promoter (CopAP) and it´s regulator CueR. CueR is a MerR like regulator, which stimulates the transcription of CopA, a P-type ATPase pump (Outten et al. 2000). CopA is the central component in obtaining copper homeostasis, it exports free copper from cytoplasm to the periplasm. This is enable by Copper induced activation of the operon transcription via CueR. The 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 (BBa_K1758320) under the control of a constitutive promoter with CopAP and sfGFP (BBa_K1758321) 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
Abdallah, A. T. and Moustafa, M. A. (2002): Accumulation of lead and cadmium in the marine prosobranchNeritisaxtilis, chemicall analysis, light and electron microscopy. Environmental Pollution, 116, 185-191.
Bergeson, L. L. (2008). "The proposed lead NAAQS: Is consideration of cost in the clean air act's future?". Environmental Quality Management 18: 79–84. :10.1002/tqem.20197 .
Borremans B., Hobman J. L., Provoost A., Brown N. L., van der Lelie D. (2001), Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstoniametallidurans CH34, J. Bacteriol., 183, 5651 – 5658
Ferner D.J., (2001)Toxicity, heavy metals.eMed J.;2(5):1
WHO Guidelines for Drinking-water Quality fourth edition, checked on 2015-09-09.
Occurrence
Mercury is found in water, typically as Methylmercury, which is build out of inorganic mercury by different marine bacteria Pseudomonas spp.) under aerobic conditions. Additional mercury(II)chloride with a high solubility, and mercury sulfide are found in water. The main natural source of Mercury exposure is trough volcanic activity (WHO 2005). Additional to the natural sources there are many kinds of emission caused by humans. For example mercury contamination can be caused by medical waste (damaged measurement instruments), fluorescent-lamps, chlor- alkali plants and thermal power plants (Verma et al.2013). The natural occurring concentration of mercury in groundwater and surface water are in most cases less than 0.5 µg/L but can rise to higher concentrations by local mineral deposit. In some countries, groundwater and shallow wells show an increased mercury concentration. Due to volcanic activity, the mercury concentration in water can rise frequently up to 5.5µg/L (Izu Oshima Island in Japan) (WHO 2005).
Health effects
In the environment, mercury is one of the most toxic elements (L.A. Rojas, 2011). Inorganic mercury is higher concentrated in water than organic mercury. The most toxic and dangerous compounds are organometallic mercury molecules like methylmercury and dimethylmercury. These organometallic compounds are better soluble in lipids. Because of this fact, it is easier to permeate the cellular membrane. Acute effects of a mercury intoxication can range from diseases of the liver, kidney, gastrointestinal tract, neuromuscular and neurological problems. Inorganic mercury accumulates in the kidneys and has a long biological half-time. In contrast to organic mercury, inorganic mercury is not able to cross the blood-brain barrier or blood-placenta barrier (Park et al., 2012). A chronic intoxication of mercury results in kidney changes, changes in the central nervous system and other effects like cancer (Holmes et al., 2009, WHO 2005). Studies have shown that mercury, in the form of mercury(II)chloride, binds with DNA and leads to strand breaks. Additional studies show that mercury generates chromosomal aberrances (WHO 2005). In addition, a relation between an early exposure of mercury and late initial of Alzheimer and other neurodegenerative diseases are discussed (Park et al., 2012).
Detection
In some laboratories mercury can be detected by atomic absorption spectrometry with a detection limit of 5µg/L and the Inductively Coupled Plasma Method with a detection level of 0.6µg/L (WHO 2005). These test systems are expensive, extensive and not suitable for fast simple testing. Additional to the named high-tech methods there are different chemical and biological test systems. One of these systems is the detection by ELISA with mercury specific Antibodies (Wylie et al. 1991).
Our mercury biosensor
For our sensor, we use parts ( BBa_K346002 and BBa_K346001) of the mercury sensor constructed by iGEM team Peking 2010. These Parts consist of the Mer operon from Shigella flexneri R100 plasmid Tn21, a mercury dependent operon. The expression of the Mer operon is regulated by the activator MerR. The MeR transcription however is regulated by itself. Mercury can bind to the C-terminal located cysteines and generates a conformal change to activate the expression (N.L. Brown et al.2003). Our mercury Sensor contains MerR, which is under control of a constitutive promoter and specific promoter MerT. sfGFP protein is used as measuring output signal and it´s transcription is controlled by the 5` untranslated region, which enhances the following reporter protein sfGFP .
References
WHO (2005): Mercury in Drinking-water Background document for development of WHO Guidelines for Drinking-water Quality checked 20.08.15
Holmes, P. ; James K.A.F.; Levy, L.S. (2009): Is low-level environmental mercury exposure of concern to human health? In SCIENCE OF THE TOTAL ENVIRONMENT 408 ( 2) pp. 171-182.
Brown, Nigel L.; Stoyanov, , Jivko V.;Kidd,Stephen P.;Hobman; Jon L. (2003): The MerR family of transcriptional regulators. In FEMS Microbiology Reviews, 27 ( 2) pp.145-163.
Rojas, LA.; Yanez, Carolina; Gonzalez, Myriam; Lobos, Soledad; Smalla, Kornelia; Seeger, Michael (2011). Characterization of the Metabolically Modified Heavy Metal-Resistant Cupriavidus metallidurans Strain MSR33 Generated for Mercury Bioremediation. PLOS ONE, 6 (3).
Verma, Rashmi; Dwivedi, Pratima (2013): Heavy metal water pollution- A case study. Recent Research in Science and Technology 2013, 5(5) pp.98-99. Park, JD.; Zheng, Wei (2012). Human exposure and health effects of inorganic and elemental mercury. In Journal Of Preventive Medicine And Public Health, 45 (6) pp. 344-352.
Wylie, Dwane E.; Carlson, Larry D.; Carlson, Randy; Wagner, Fred W.; Schuster, Sheldon M. (1991). Detection of mercuric ions in water by ELISA with a mercury-specific antibody. Analytical Biochemistry, 194 (2) pp. 381-387.
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.
ISO, 1996 US: Technical Factsheet on: Nickel, checked on 9/9/2015.
