The development of the biosensor is central to the success of BioSensei, a modular approach for its development has been followed:
• A chassis microbe – the host bacterial species for the other units
• Sensing units– identifies the presence of target pollutants
• Processing units – produces signalling molecules upon a trigger
• Calibration units – mediates the expression of a processing unit under the control of a sensing unit
This has allowed for the simultaneous development and evaluation of sensing and processing units, using established interim units. Pseudomonas. Putida EM42, a commonly used non-motile strain of a non-pathogenic bacterium, was selected as the chassis microbe due to its documented previous success as a chassis microbe, its pollution resistance and survivability in river water.
Several approaches to develop sensing units have been trialled, including:
• implementation of established interim sensing units (salicylic acid, rhamnose)
• incorporating known systems from other bacterial species
• generating new systems based on known systems
• use of native interactions with target pollutants
• intracellular signals
So far, BioSensei has successfully implemented a Nitrate sensing unit based on an E. Coli system.
Several processing units have been developed, including:
• regulation of native production of pyoverdine (PVD) in P. Putida
• transfer of the production pathway of pyocyanin (PYO) from Pseudomonas Aeruginosa
• production of fluorescent proteins, e.g. green fluorescent protein (GFP)
• transfer of the production pathway of pyochelin (PCH) from P. Aeruginosa (under development)
These molecules are good candidates form chemical signals as they are all optically and/or electrochemically detectable.
Initial demonstrations of the biosensor were achieved in P. Putida, utilising the system modularity and well-established sensing/processing units, in conjunction with the developed systems yielding biosensors with:
• PVD induced by rhamnose
• PYO induced by salicylic acid
• GFP induced by rhamnose
• GFP induced by nitrate
• PVD induced by nitrate
The PVD production, induced by the nitrate sensing unit, demonstrates the successful BioSensei biosensor concept. BioSensei will continue to target new sensing units during its second half, to address more of its target pollutants.
Biosafety is an essential aspect of BioSensei, following all biological safety best practices. An additional hierarchical containment strategy is under development, involving:
• Use of a non-motile chassis bacterium
• Multiple layers of polymer encapsulation
• semi-porous membranes to prevent biosensor escape
The containment development has been based guided by simulations and experimental validation of their effectiveness without compromising the function of the sensor system.
Optical and electrochemical transducers have been codeveloped to convert the chemical signals into electrical signals. They have been successfully tested with the biosensor/its output with rhamnose induced GFP and salicylic acid induced PYO for the optical and electrochemical transducer respectively.
The transducers will be controlled by supporting electronics including a bespoke electrochemical analog front end, a control board, a machine learning processor, and communication equipment, which have been designed awaiting production and testing. They will collect, collate and interpret the signals and transmit them securely over the internet to the end-user virtual dashboard for interpretation in near-real time.
