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Policy & Practices
We have developed Clickable Outer Membrane Biosensors as a universal membrane sensor platform for biosensors. Our dream is that any chassis may be functionalized with COMBs. This chassis should then be able to detect virtually any biomarker and translate detection of this biomarker into any desired response. To obtain such a system, it is of the utmost importance that the system is inherently modular. The modularity of our system is reached through rational choices for the recognition element, the scaffold and the signaling components for our COMBs.
The recognition elements - Aptamers
Molecular recognition is central to biosensing [1]. The moieties responsible for molecular recognition are better known as recognition elements. Recognition elements frequently used in biosensors are aptamers. These aptamers are small oligonucleotides which fold spontaneously into intricate three-dimensional structures. These structures fit perfectly into a wide range of disease markers, with affinities reaching into the low nanomolar range. Through modifications, these aptamers can be attached post-translationally to our proteins, resulting in a truly modular membrane sensor platform. Read more on aptamers:
Aptamers are folded oligo- nucleotides (orange) which perfectly fit into biomarkers (purple).
Image credit: Flanders' FOOD
Image credit: Flanders' FOOD
The scaffold - Outer Membrane Proteins
Outer membrane proteins form an essential component for Gram-negative bacteria, providing the bacteria with protection against a harsh environment [2]. More than six outer membrane protein families have been discovered, which all share a beta barrel secondary structure. Thanks to this structure, the proteins feature loops protruding from the bacterial outer membrane, and intracellular C-termini. This enables clicking of aptamers to the membrane proteins and fusing signaling components to the outer membrane proteins intracellularly, making outer membrane proteins the perfect scaffold for COMBs. Read more on outer membrane proteins:
OMPs can feature as a scaffold as aptamers can be clicked on the protruding loops & signaling components fused intracellularly.
The signaling components
Molecular recognition in our device Fis translated into close proximity of two membrane proteins. The signaling components used for COMBs translate a close proximity into a response. Signaling components which do exactly this are split luciferases & fluorescent and bioluminescent proteins, through Resonance Energy Transfer. In the future, TEV Proteases may be used to translate a proximity into the release of the transcription factor, coupling COMBs recognition to the vast amount of cellular responses bacteria have available. Read more our signaling components:
COMBs signaling components translate a close proximity into a measurable signal. Here shown is BRET.
General Outline
To evaluate the utility of the E.coli sensor three different scenarios will be scrutinized. The E.coli sensor system could possibly be used for the detection of diseases related to disturbances in the intestinal tract. Furthermore we will examine the use of our sensor system to detect pathogens related to Q-fever, a societal problem in the Netherlands. Finally we will look at the applicability of the sensor system to reduce the use of pesticides.
For each of these scenarios we have looked at law and legislation, ethical aspects and the vision of different stakeholders upon the problem and our proposed solution.
Law and legislation is a critical factor when it comes to bringing a product to the market. Different instances are concerned with testing the safety of a product before it reaches the consumer. One of the important instances in the Netherlands that we contacted was the RIVM. In Europe the EMA is an important regulator, responsible for the scientific evaluation of medicines developed by pharmaceutical companies.
Ethical aspects play an important role in the general attitude of the population towards new technological ideas and developments. To introduce these new possibilities within the field of synthetic biology to society, we are giving presentations and have ethical debates with them about the risks, consequences and possibilities. For the ethical aspects around our applications we contacted the CCMO, an institute in the Netherlands that is responsible for performing ethical test on developed medical treatments.
Other stakeholders we contacted are research institutes with regards to the intestinal tract, Q-fever and pesticides. Gastroenterologists and patients have been approached for the scenario about diseases within the intestinal tract. For the scenario about Q-fever we contacted infected farms and for the scenario about pesticides we contacted nurseries who are dealing with the overuse of pesticides.