Occurrence

Arsenic is found in nature in both organic and inorganic forms, typically as arsenite (As^III) or arsenate (As^V). 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. As^III can bind to three cysteine residues in ArsR. The resulting conformational change deactivates the repressor (Chen, Rosen 2014).

References

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 (Cr^III) and the hexavalent (Cr^VI) (Mitchell D. Cohen et al.).

Health effects

While the trivalent form is an essential dietary mineral and the most common natural form, it is of interest to detect 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). Its 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, where it results in oxidative stress reactions (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 of 120 µg/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 an iodide reaction for measurement (GIORGIA).

Our chromium biosensor

We work with the chromate inducible operon of Ochrobactrum triti ci5bvl1, which enables a tolerance for chromium VI and superoxide. The expression of the operon depends on the bondage of the repressor chrB to the operator sequence (Branco et al. 2008). The chrBACF operon (BBa_K1058007) and the chrB repressor were introduced by the team BIT 2013.

References

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 through the liver. It is used in coating 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 guidelines for drinking water the maximal concentration of copper is 2 mg/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 manifests in a misfunction, so that copper can not be excreted by the liver into bile. If not treated this can lead to brain and liver damage (US EPA ORD NCEA Integrated Risk Information System (IRIS) 2014). Some studies associate high copper levels with aging diseases such as atherosclerosis and Alzheimer’s disease (Brewer 2012).

Detection

The most important analytical method for the detection of copper in water is the inductively coupled plasma mass spectrometry (ICP-MS),which has the lowest detection limit (0.02 μg/L). Other methods often used for detection are the atomic absorption spectrometry (AAS) with flame detection, which has the highest (20 μg/L) as well as graphite furnace atomic absorption 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 copper homeostasis from E. coli K12. This includes the promoter (CopAP) and its 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 enabled 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). We combined CueR (BBa_K1758320) under the control of a constitutive promoter with the operator site of CopA Promoter and sfGFP (BBa_K1758321) for measuring output signals.

References

Occurrence

Lead is a heavy metal with widespread occurrence. The relatively simple extraction methods and several desirable properties have made it useful to humans. Lead and lead compounds are used in a high variety of products, such as pipes and plumbing materials, solders, gasoline, batteries, ammunition and cosmetics. Therefore, lead plays a major role in the industry and is one of the most used metals. Compared to other metals its occurrence is relative low. Lead can be detected in different parts of the environment, like air, soil and water. A high concentration of lead in drinking water is often induced by obstruct pipes that consist of lead or that has a part of lead, respectively. This allow water to be easily contaminated. That’s often a problem in houses where lead is used in household plumbing. Due to the fact that lead is also occurring in water, it could results in adverse health effects (WHO: Fact sheet number 379, Lead poisoning and health).

Health Effects

Lead has no biological role in the body, but is a highly poisonous metal. The ingestion of lead could affect almost every organ and system in the body (EPA Health Effects: How Lead Affects the body). The main target for lead toxicity is the nervous system. It can have acute or chronic health effects. The acute health effects are occurring immediately after contact with lead. This can be irritation of the eyes or can cause headache, irritability, disturbed sleep, and mood as well as personality changes. Exposure to higher lead concentrations over a long-term could cause serious damage to the brain and to the kidneys. And finally the damage can 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, L., 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 a fast review of the water quality is a worthwhile aim. The method for detection is currently based on the principle of flame atomic absorption spectrometry (FAAS) (Abdallah, A. T. and Moustafa, M. A., 2002). This detection method is difficult to implement in developing countries, and time consuming thus there are hardly any quality standards.

Our lead biosensor

For our biosensor we use parts of the chromosomal lead operon of Cupriavidus metallidurans. Naturally the operon for lead resistance contains pbrT, which encodes a Pb(II) uptake protein, pbrA, which encodes a P-type Pb(II) efflux ATPase, pbrB, which encodes a predicted integral membrane protein and pbrC, which encodes a predicted prolipoprotein signal peptidase. The expression of the operon is regulated by the repressor pbrR. As a MerR like regulator, its stimulates the transcription of the operon in the presence od Pb²⁺ Borremans et al., 2001). Our sensor system combines pbrRunder the control of a constitutive promoter and the PbrA promoter for the lead depending expression of sfGFP.

References

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 through volcanic activity (WHO 2005). Additional there are many kinds of emission caused by humans. For example mercury contamination can be caused by medical waste (damaged measurement instruments), fluorescent-lamps, chloralkali plants and thermal power plants (Verma et al., 2013). The natural occurring concentration of mercury in groundwater and surface water are in general less than 0.5 µg/L but can rise to higher concentrations by local mineral deposit. Due to volcanic activity, the mercury concentration in water can rise frequently up to 5.5 µg/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). The most toxic compounds are organometallic mercury molecules like methylmercury and dimethylmercury, bceause they can easly permeate the cellular memrbane. 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, before it is not detectable anymore. In contrast to organic mercury, inorganic mercury is not able to cross the blood-brain barrier or blood-placenta barrier, so it accumulate in the organics (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). 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

Mercury can be detected by atomic absorption spectrometry with a detection limit of 5 µg/L and the Inductively Coupled Plasma Method with a detection level of 0.6 µg/L (WHO 2005). 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 the Mer operator from Shigella flexneri R100 plasmid Tn21 ( BBa_K346002 ) and its regulator MerR ( BBa_K346001 ) constructed by the iGEM team Peking 2010. The MerR functions as an activator and regulates his own transcription by binding to his C-terminal cysteine resulting in a changed conformation (N.L. Brown et al., 2003). Our sensor system combines merR under the control of a constitutive promoter and the MerT promoter for the mercury depending expression of sfGFP.

References

Occurrence

The amount of natural occurring nickel in comparison to other heavy metals is quite low even if it is an element 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 through releases from taps and fittings the nickel may accumulate to concentrations up to 1 mg/L. In special cases of release from natural or industrial nickel deposits in the ground, there may be higher concentrations in drinking-water. Through unintended release the concentration can be higher than the guideline value of 0.07 mg/L (WHO: Guidelines for Drinking-water Quality, Fourth Edition)

Health effects

Even though nickel is essential for mammals and a part of human nutrition, it may cause dermatitis as well as itching of fingers, hands and forearms by 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.

Detection

The two most commonly used analytical methods for nickel in water are atomic absorption spectrometry and inductively coupled plasma atomic emission spectrometry. Inductively coupled plasma atomic emission spectroscopy is used for the determination of nickel detection limit of about 10 μg/L (ISO, 1996). Flame atomic absorption spectrometry is suitable in the range of 0.5–100 μg/L (ISO, 1986). A limit of detection of 0.1 μg/L can be achieved using inductively coupled plasma mass spectrometry. Alternatively, electrothermal atomic absorption spectrometry can be used. (cavillona 2005)

Our nickel biosensor

For our nickel sensor system we used the rcn-operon from E. coli which encodes a nickel- and cobalt-efflux system with a high sensitivity to nickel. In the absence of Nickel the respressor RcnR regonizes the operator site and blocks transcription of the operon, while in the presence of nickel it is abadoned due to a conformational change. Our sensor system combines rcnR under the control of a constitutive promoter and the prcnA promoter for the nickel depending expression of sfGFP. If Ni²⁺-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<>/i transcription. (EPA et al., 2013; Blaha et al., 2011; Iwig et al., 2006) Our output signal works through fluorescence.