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Team:Tuebingen/Implementation

PacMn

Implementation

Introduction

We are happy to be able to contribute to the iGEM knowledge base with our work and the data we collected. But for our technology to have an impact for a broader community outside of this competition, there is still much work to be done. The following paragraphs will give insight into the considerations that we made regarding possible real-life applications of our biosensor.

Who can use our technology?

When considering possible real-world applications of our Mn-biosensor, it is useful to start at the main sources of manganese pollution (1).
  • Mining of manganese-rich ores
  • Steel production – especially Mn-rich alloys
  • Disposal of sewage sludge from wastewater treatment facilities

There are four main user groups that must be considered:
  1. Private firms active in manganese mining and processing
  2. Municipal wastewater treatment facilities
  3. The scientific community and regulatory bodies interested in the effects of Mn-species
  4. Consumers concerned about the quality of their drinking water

For every user group, there are specific requirements that must be met when designing a product targeted at a given group. For example, the consumer biosensor must be widely available and usable without any lab-equipment. It should also be easy to use without much scientific background knowledge. Concerning these points a cell-free solution would likely be most successful. On the other hand, for scientific users, reliable calibration and quantitative as well as quantification measurements are essential. Here, we intentionally do not limit our considerations to one of these possible use-cases, but as we provide a basic proof-of-principle in our result section, we want to give an overview on multiple possible products that can be developed from our biosensor.

How can our technology be improved in the future?

Due to the clear-cut timeframe of the iGEM-competition, it is only natural that a project of this calibre will not proceed past the initial stages of proof-of-concept and lab-testing, especially in the times of a global pandemic. However, we propose several improvements of our system that alone or in combination would lead to a more competitive product:
  • Combination of the sensing capabilities with other heavy-metal-elements, such as cadmium or lead, the first of which already has its own biosensor system based on E. coli established by the iGEM Team York in 2014 (2).
  • Development of hardware to make the system applicable in a non-lab environment, e.g. a hollow-fibre-bioreactor for continuous monitoring of Mn-levels in wastewater treatment facilities. Testing of a hollow-fibre-reactor for the removal of heavy metal elements was done by the iGEM Team Cornell in 2015 (3).
  • Improvement of the Mn-binding capabilities of the system by engineering the phytochelatin to a higher binding constant.
  • Transfer the sensor system to a cell-free application (4), e.g. a dip-stick-solution for rapid and low-tech applications in lesser developed regions such as proposed by the iGEM Team Bielefeld in 2015 (5).

What risks for the safety of humans and the environment could arise from the application of our technology?

When assessing the risks of our Mn-biosensor, it is important to always weigh potential risks with the benefits that arise from using the system. Manganese, due to its long biological half-life, can accumulate in the brain even when environmental levels are not exceptionally high (1). Raised Mn-levels in drinking water have been linked to decreased intellectual function and higher incidence of neurodegenerative diseases in children in multiple studies (6,7).
As the E. coli bacteria used in our project are commonly used in wastewater treatment facilities and are also not pathogenic for humans, animals or plants, there is generally little risk of harming humans or the environment. However, since our bacteria are genetically modified organisms (GMOs), special precautions are mandated by national legislation, e.g. in Germany it is only legal to work with genetically engineered strains in officially registered laboratories, provided that no official admission for the release of the given GMO was granted.

Are there any other challenges that hinder useful applications?

Concentrations of Mn (II)-species in water that is not directly contaminated by industrial sources of Manganese are typically only a few µg/l (8). To precisely measure Mn-concentrations in that order of magnitude, our biosensor would need further improvements to have a higher sensitivity and a better fluorescent response. This could be faced by improving the regulating sequences in the constructs and modelling the Manganese binding proteins (Phytochelatin) to receive a higher binding affinity.
Furthermore, truly quantitative measurements with our system remain a major challenge, since it would also have to be verified that the sensitivity of our biosensor does not change over time, e.g. due to the bacteria developing a higher tolerance for Mn. Manganese could be toxic in higher concentrations or at least lead to cell stress, what could affect stable expression of the detection constructs. But also, other factors like pH, salt concentrations, and temperature have an influence on the strength of genetically encoded fluorophores like FAST2, which further complicates analysis of environmental samples.
Another point is to reduce the costs of a possible application, to reach a broader range of usage fields. Since there are already Manganese tests available, e.g. based of measuring absorbance due to a color change (e.g. 10,11), our test has to be optimized in regard of cost and other benefits e.g. measure the concentration of different heavy metal ions in one test or a higher sensitivity. Especially the cost factor has to be optimized in a later stage of the project.
Strict GMO-legislation, especially in European countries, also plays a key role in the success of our biosensor system. In Germany, the admission of new GMO´s is regulated on a case-by-case method (9), i.e. in the current state of the project, no prediction can be made whether a final product would comply with the regulations or not. However, cell free applications are most often not governed by GMO-law, so further development in this direction can expect less complication in terms of legal issues.

References