Development of a light driven biohybrid microbot

Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Its presence is crucial for human health as it aids in the absorption of nutrients. Growing in aerobic and anaerobic environments, it is highly adaptable and a versatile model organism for different scientific studies. E. coli has been extensively studied in molecular biology, genetics, and biotech- nology due to its relatively simple genome, rapid growth rate, and the ease with which it can be manipulated genetically. It was one of the first organisms to have its genome sequenced, and this accomplishment has paved the way for numerous advances in genetic engineering and synthetic biology. The bacterium’s ability to express foreign genes has been harnessed to produce proteins, enzymes, and other biological compounds. An example of this is being used for the production of insulin and bringing down its cost by a huge factor. In a way, it acts like a biological circuit board, where one can hack into its programming and modifying its properties and behaviour. This also makes it a good candidate to study physical phenomena. This thesis explores the development, optimization, and applications of microbots that are powered and controlled by such genetically modified E. coli bacteria. The important gene modification we do makes the bacteria sensitive to green light, and through this we can gain control over its speed. By integrating biological elements with micro-robotics we investigate different complex phenomena. The research is divided into two main parts: the theoretical foundations and practical applications of microbots, and the methodologies for their fabrication and use in bacterial baths. The first part of this thesis is regarding the microbot. In the first chapter, we get an introduction to the E. coli bacteria, the hydrodynamics of flagellar motion, the gene modification done to make these bacteria and the study of proteorhodopsin, the bacterial light switch. We take an overview of the current trends and principles of microbotics as well as scaling laws of the physical forces on a micron scale and their limitations, framing key research questions guiding this study. The second chapter, focuses on the development and optimization of the microbot design, including the two-photon fabrication parameters and calibration of bacterial chambers. We take a look at the measurement of drag coefficients and the effects of connector length on a two-propeller design. In chapter 3, we briefly describe the methods used in our studies and present results about the microbot behaviour under uniform illumination and dynamic feedback conditions. We discuss the Development of tracking and control algorithms to enhance microbot efficiency. Chapter 4 focuses applications of microbots for transporting cargo beads within microfluidic environments and to study geometric optics approximation in self-propelled particles. This chapter also has sections on other projects that have been carried out in the duration of this thesis. These include the investigation of using 3D microstructures, including active pressure around curved boundaries and torque measurement with light mills. Part 2 of this thesis focuses on the methodologies used in the development of microbots. Chapter 5 is focuses on the two-photon polymerization. This is a versatile tool that can be used in rapid prototype development of 3D structures in micron and sub-micron scale. We also go through the protocols for sample preparation and microbot post-processing that were developed in the course of this study. Chapter 6 explains the techniques used for bacterial cell culture and investigation of bacterial speed using differential dynamic microscopy. In the course of this thesis, I developed a unique micro-robotic system that integrates bacteria into synthetic 3D microstructures to extract mechanical work. I designed and 3D fabricated the synthetic chassis using the two photon polymerization process, calibrated it for the bacterial cells, developed coating protocols to reduce surface interactions and develop a code to analyze and control the trajectories of multiple microbots continuously captured through the microscope.This system can control individual microbots autonomously and required a multi-disciplinary approach, from research topics in physics, microbiology, synthetic biology, material sciences, robotics and control theory and computer science. This system can find applications in different fields of study.