Rescue missions are dangerous operations in which fast decision-making can make the difference between failure or success in saving human lives. Biosensors are simple yet highly selective tools for measuring chemical signals, which can be crucial in these situations. However, state-of-the-art cell-based biosensors rely on slow-acting gene regulatory networks and are difficult to read out. Therefore, we are excited to take on the challenge of designing a biosensor that senses compounds within a time scale of seconds by exploiting post-translational processes. To illustrate the potential of our system, we will then integrate our sensor on a mobile robot designed to sense chemicals such as explosives and toxins.
In this way, we will combine the best of both worlds: biology will provide the specificity and sensitivity, while electronics will provide a fast interface.
The biological core of the project consists of the acceleration of the sensing of chemical ligands. We will approach the problem with two different strategies based on two different biological processes.
a) Bacterial motility can be influenced within a very short time
Chemotaxis is described as the directed movement of a cell along a concentration gradient of a chemical species. Chemotaxis is usually executed through the flagella and a molecular sensing apparatus that allows the bacterium to give a specific movement. Overall, directionality is achieved by integrating over two types of movements: swimming movements to rapidly advance towards the source of the ligand or tumbling to change direction away from it. The balance between these two behaviors is determined by the amount of ligand detected by the bacteria. Therefore, by sensing changes in movement patterns of bacteria, one could determine, and even quantify, the presence of a certain chemical compound in the bacterial surroundings within seconds. The ability of bacteria to sense chemicals resides in a wide variety of membrane receptors with a modular structure that can be engineered to respond to a variety of different ligands, opening the door for the almost instantaneous bio-sensing of multiple molecules of interest.
Cyclic di-GMP (c-di-GMP) is a second messenger that integrates many extracellular and intracellular signals in different bacterial species, including regulating bacterial swimming speed in P. putida. Surface receptors containing cytoplasmic effector domains can regulate the intracellular levels of c-di-GMP in a matter of seconds. We propose that we can engineer surface receptors to change intracellular c-di-GMP levels in response to specific ligands, and read out such changes by optically recording changes in the average swimming velocity.
b) Using DNA-binding transcription factors to generate fast signals
Cellular responses to environmental cues are often transcriptional, meaning that the response to the presence of an external signal is mediated by the conditional binding of a transcription factor (TF) to a specific sequence in the DNA, which in turn promotes the expression of specific genes. This takes time, usually in the range of tens of minutes or even hours, a timescale often not useful for our biosensing purposes. We propose to accelerate this process by bypassing the process of transcription and translation. Instead, ligand-induced operator binding of a fusion protein consisting of the sensing and the DNA-binding domains of the TF and one half of a split reporter protein, complements the second part of the reporter protein which is directed to the correct place on the DNA by fusion to a second DNA-binding protein.
We propose to showcase the power of accelerated biological sensing by using it to direct the movement of a sensing robot. To this end, we will first construct a microfluidic sensing device. Microfluidic techniques offer the possibility to automate steps, while providing faster response times, enhanced analytical sensitivity and portability.
We plan to implement a microfluidic-based system to sample volatile molecules and convey them to the sensing bacteria kept within the microfluidic setup. The crucial part will then consist of a fast and compact imaging apparatus to translate the movement output from the bacteria into an actionable signal for the robot. We propose to do that by letting our robot explore the environment, sampling the surrounding air at the current position, directly conducting it to the microfluidic device, receiving the response from the bacteria, comparing it with previous measurements, and then adjusting its movement trajectory according to the information gathered. The optical readout will be implemented via a lensless imaging system capable of tracking the movement properties of a bacterial population.