The Problem

Drinking water is the most basic of all resources, but its global exposure to toxic substances is a big problem. In particular, heavy metals endanger the health of millions of people. These metals occur naturally in small amounts in the earth’s crust. However, by erosion or human activities, such as mining or agricultures, they are released and can accumulate in soils and waters. That is why the groundwater in many regions of the world is contaminated for example with arsenic. Among others India and Bangladesh are particularly affected by this issue. But even in countries where the supply with clean water is a minor problem, heavy metal contaminations may occur as well, for example caused by old lead pipes. Since these last meters of water transport normally are not controlled, such contaminations often remain undetected for a quite long time.

While some heavy metals are essential as trace elements, others lead to health consequences already in low concentrations. But in any case, the intake of larger amounts of heavy metals entails a variety of serious diseases such as nerve damage, cancer and even can lead to death. Therefore it is essential to control the heavy metal content of water and food. To enable this, a simple favorable and at the same time secure analytics is required. As laboratory tests and conventional test kits are in general not suitable for the public, we decided to establish a biosensor for various heavy metals.

The second topic we want to deal with is motivated by the fact that recently crimes in which so-called knock-out-drugs (date-rape-drugs) were used become more frequent in our region. These drugs are usually a mixture of different substances which produce narcotic effects and thus make the victim defenseless and unable to act. By now the detection of knock-out-drugs is only possible in retrospect in the laboratory and this just in a limited time frame. However there is no opportunity for checking suspicious drinks quickly and easily. We want to change this by developing a biosensor for the detection of knock-out-drugs.

The Detection System

Many organisms have genes that make them resistant to toxic substances or enable the use of particular nutrients. However, these genes are only required if the relevant substance is actually available. A permanent expression would be an unnecessary waste of energy for the organism. Therefore, control mechanisms have evolved ensuring the identification of specific substances to allow expression of certain genes only in presence of this substance.

The lac-operon which is required for the metabolism of lactose in Escherichia coli and many other bacteria is a classic example for such a mechanism. In absence of lactose, a repressor protein binds to the operator site of the lac-operon thereby obstructing the binding of the RNA polymerase to the promoter. Consequently there is no mRNA transcript of the following lac-genes. As the repressor is an allosteric protein it may assume two different shapes. In presence of lactose the inducer allolactose binds to the repressor triggering a conformational change. As a result the repressor releases the operator DNA and thus allows the RNA polymerase to bind to the promotor and express the genes.

In some bacteria similar systems enable the resistance to certain heavy metals. For instance there are bacteria which are able to grow in presence of arsenic, because they have a mechanism for carrying the heavy metal out of the cell. The required pump for this process is only formed when a specific repressor was previously inactivated by the binding of arsenic.

These natural mechanisms can be used to construct biosensors. Therefore, the genes that are naturally activated in the presence of a particular substance are replaced by a reporter gene. If this reporter gene is expressed, it provides a measurable signal. This signal may be, for example, a color change or a fluorescence signal.

Such biosensors allow a highly specific detection of minute amounts of a substance. Moreover, biosensors can be produced inexpensively whereby they are in particular of interest to developing countries.

Our Biosensor

Although, researchers are already working for many years on biosensors, these sensors have not yet arrived in everyday life. This can be explained with the fact that sensors based on living microorganisms create a number of problems. This involves on the one hand legal and security issues - since working with genetically modified organisms, their release into the environment must be prevented. On the other hand, these sensors are often not very user-friendly, as living cells have a limited suitability for storage and the application outside the laboratory is complicated. In order to solve these problems and thus make biosensors applicable in everyday life, we are working on two cell free systems for our biosensor.

Our first concept is based on the in vitro transcription and translation including protein biosynthesis outside of a living organism. It is already known that there is the possibility to run this central process in a cell extract. For this purpose the cells are disrupted and an energy-source as well as other supplements is added. This concept of cell-free protein synthesis (CFPS) is beneficial for example when a protein has to be produced which is toxic for the living cell.

Recently, it has been shown that it is possible to make the cell extract storable for a long time by freeze-drying it on paper (Pardee et al., 2014). With this freeze-dried cell extract protein synthesis was even possible after a long-term storage at room temperature only by adding water. In our opinion, this system is very promising for the development of easy-to-handle biosensors. Currently, we are working on the production of a cell extract and we intend to develop a test strip for heavy metals and knock-out drugs.

In parallel, we are working on a second concept that works with isolated proteins and DNA. This system is based on the interaction between a repressor protein and DNA. Thereby the repressor is immobilized on a surface and binds a small DNA molecule that carries its recognition sequence. Now, adding a water sample that contains the corresponding inducer causes a loss of the repressor-DNA-interaction and the DNA becomes detached. If this detaching is combined with the loss or generation of a signal, it is possible to detect whether the sample contains a certain substance or not.
The advantages of this system are its simplicity and thus its potential robustness. Furthermore, it works completely cell free and is therefore very interesting regarding biosafety.