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PacMn - Phytochelatin-actuated complexation of manganese

Due to the industrial revolution, for most people the standard of living drastically increased in the last 150 years. On the one hand this resulted in an increase of life expectancy, economic prosperity and improved access to medical care. On the other hand, the development was enabled through the carbon dioxide emitting exploitation of fossil resources eliciting a gradually increasing burden on our environment.
As a consequence, next to greenhouse gas (like carbon dioxide) emissions and the accumulation of microplastic in the oceans, the advancing destruction of ecological and economical valuable soil is a major problem of the 21st century. The catastrophic global impact of these developments is broadly demonstrated by the drastic increase of environmental disasters such as droughts, which is also reflected by the rising numbers of iGEM projects from all around the world, addressing environmental issues.
An important issue which has re-emerged in scientists’ and environmental organizations’ focus is heavy metal pollution of the environment. The anthropogenic release of heavy metals leads to their accumulation in the soil. Here, they are taken up by organisms, including plants, entering the food chain and finally ending up on our plates, thus posing a threat not only to animals but also us humans.
Detecting heavy metals using chemical testing is often very laborious and expensive, being conducted in specialized laboratories. Therefore, especially structurally weak regions may not have the respective infrastructure for preventive testing. Accordingly, the development of more versatile and economic options, such as biosensors that use biological compounds and organisms for substance detection, has become a major research focus of synthetic biology.
The rapidly evolving field of synthetic biology and the opportunities proposed by an increasing genetic toolbox as well as the possibility of providing a product which may improve the standard of living for many people has inspired us to develop PacMn, a biosensor for the detection and chelation of manganese.

Why manganese?

Despite the successful reduction of acute manganese poisoning at the workplace with the instatement of workplace security measures, the recent development towards rising chronic manganese poisoning caused by continuous exposure is very alarming (1). The consequences of manganese poisoning severely attack the nervous system, causing Manganism, a Parkinson-like disease. Thus, it is of great importance to reduce chronic manganese exposure (1).
With 0.05mg/l the World Health Organisation (WHO) has determined a limit for manganese in drinking water. However, especially developing countries like Bangladesh, this threshold is often exceeded in more than 50 % of the area (2). Via bioaccumulation, manganese accumulates in crops like rice, wheat and tea and life-stock and fish, respectively (3). As these are basic foods for the majority of the world’s population, chronic poisoning with manganese is a serious threat which should be eliminated.
Next to the demand for inexpensive alternatives for chemical manganese testing and the global threat posed by the heavy metal, we were further inspired by the discovery that we are also locally affected by manganese bioaccumulation. Due to the climate-change triggered increase in groundwater temperatures, many regions in Germany have shown a measurable increase in manganese in groundwater resources, posing a threat to the environment and the German public (4). Although the underlying mechanisms have not been fully elucidated, this further supports the importance of research for heavy metal detection and clearance systems.

PacMn - Phytochelatin-actuated complexation of manganese

Although there are highly specific heavy metal detection systems available, there is still a demand for more economic but equally efficient detection methods. Thus, we want to utilize the vast possibilities proposed by synthetic biology applications for the design of a simple but robust bifunctional biosensor which not only detects manganese but also clears the environment of the pollutant.
In accordance with this, in this year’s iGEM project, to detect manganese we introduced a manganese sensing riboswitch and a fluorescent FAST-2-tagged reporter protein into E. coli using classical methods of synthetic biology. Under the control of either a constitutive anderson promoter the FAST-tagged protein is continuously transcribed, however only translated upon the interaction of a manganese with the manganese riboswitch located upstream in the untranslated region (UTR) of the protein. Via the binding of manganese to this riboswitch, a conformational change is triggered and the ribosomal binding site (RBS) is released, inducing translation of the FAST-2-tagged reporter. We chose FAST-2 due to various reasons, namely its versatility and, unlike many established fluorescent proteins such as GFP, putative application under anaerobic conditions. Furthermore, fluorescence is only detected upon the addition of a fluorophore, which may be exchanged, allowing for measurements of different wavelengths.
As our bifunctional biosensor should not only sense but also clear manganese, we have additionally cloned a gene for FAST-2-tagged synthetic phytochelatin into our biosensor sequence. Phytochelatins are heavy metal detoxifying oligopeptides originating from plants. Via their chelation of manganese, they can presumably be exploited for the clearance of manganese from the environment.
To conclude, a smart combination of the above mentioned biological modules will be an innovative contribution to recent research in synthetic biology and metabolic engineering. Furthermore, following the iGEM tradition, we propose an explicit application with immediate improvement on the quality of life of many people, regionally and world-wide.