Mobilization of Pollutant-Degrading Bacteria by Eukaryotic Zoospores

This study was supported by the Spanish Ministry of Science and Innovation (CGL2010-22068-C02-01 and CGL2013- 44554-R), the Andalusian Government (RNM 2337), and the CSIC JAE Program (RS). PvW has funding support from the BBSRC and NERC. Thanks are also given to Sara Hosseini of the Uppsala BioCenter, SLU, Uppsala, Sweden for a useful discussion on oomycete zoospores.Peer reviewedPostprin

Bacterial Biohybrid Microswimmers

Over millions of years, Nature has optimized the motion of biological systems at the micro and nanoscales. Motor proteins to motile single cells have managed to overcome Brownian motion and solve several challenges that arise at low Reynolds numbers. In this review, we will briefly describe naturally motile systems and their strategies to move, starting with a general introduction that surveys a broad range of developments, followed by an overview about the physical laws and parameters that govern and limit motion at the microscale. We characterize some of the classes of biological microswimmers that have arisen in the course of evolution, as well as the hybrid structures that have been constructed based on these, ranging from Montemagno's ATPase motor to the SpermBot. Thereafter, we maintain our focus on bacteria and their biohybrids. We introduce the inherent properties of bacteria as a natural microswimmer and explain the different principles bacteria use for their motion. We then elucidate different strategies that have been employed for the coupling of a variety of artificial microobjects to the bacterial surface, and evaluate the different effects the coupled objects have on the motion of the 'biohybrid.' Concluding, we give a short overview and a realistic evaluation of proposed applications in the field

Biohybrid robotics: From the nanoscale to the macroscale

Biohybrid robotics is a field in which biological entities are combined with artificial materials in order to obtain improved performance or features that are difficult to mimic with hand-made materials. Three main level of integration can be envisioned depending on the complexity of the biological entity, ranging from the nanoscale to the macroscale. At the nanoscale, enzymes that catalyze biocompatible reactions can be used as power sources for self-propelled nanoparticles of different geometries and compositions, obtaining rather interesting active matter systems that acquire importance in the biomedical field as drug delivery systems. At the microscale, single enzymes are substituted by complete cells, such as bacteria or spermatozoa, whose self-propelling capabilities can be used to transport cargo and can also be used as drug delivery systems, for in vitro fertilization practices or for biofilm removal. Finally, at the macroscale, the combinations of millions of cells forming tissues can be used to power biorobotic devices or bioactuators by using muscle cells. Both cardiac and skeletal muscle tissue have been part of remarkable examples of untethered biorobots that can crawl or swim due to the contractions of the tissue and current developments aim at the integration of several types of tissue to obtain more realistic biomimetic devices, which could lead to the next generation of hybrid robotics. Tethered bioactuators, however, result in excellent candidates for tissue models for drug screening purposes or the study of muscle myopathies due to their three-dimensional architecture

Microfluidics Expanding the Frontiers of Microbial Ecology

Microfluidics has significantly contributed to the expansion of the frontiers of microbial ecology over the past decade by allowing researchers to observe the behaviors of microbes in highly controlled microenvironments, across scales from a single cell to mixed communities. Spatially and temporally varying distributions of organisms and chemical cues that mimic natural microbial habitats can now be established by exploiting physics at the micrometer scale and by incorporating structures with specific geometries and materials. In this article, we review applications of microfluidics that have resulted in insightful discoveries on fundamental aspects of microbial life, ranging from growth and sensing to cell-cell interactions and population dynamics. We anticipate that this flexible multidisciplinary technology will continue to facilitate discoveries regarding the ecology of microorganisms and help uncover strategies to control microbial processes such as biofilm formation and antibiotic resistance.National Science Foundation (U.S.) (Grant OCE-0744641-CAREER)National Science Foundation (U.S.) (Grant IOS-1120200)National Science Foundation (U.S.) (Grant CBET-1066566)National Science Foundation (U.S.) (Grant CBET-0966000)National Institutes of Health (U.S.) (NIH grant 1R01GM100473-0)Human Frontier Science Program (Strasbourg, France)Human Frontier Science Program (Strasbourg, France) (award RGY0089)Gordon and Betty Moore Foundation (Microbial Initiative Investigator Award

Antigen 43-mediated biotin display and fabrication of bacteria-driven microswimmers

Controlled attachment of bacterial cells to biotic and abiotic surfaces without affecting their fitness is of great interest in biotechnological applications, such as patterning surfaces with cell-based biosensors, cell-cell attachment in syntrophic communities and fabrication of bacteria-driven biohybrid microswimmers. For years genetically modified outer membrane proteins and autotransporters were used to functionalize the bacterial cell surface with peptides and small proteins used for peptide library screening, bioremediation and biocatalysis. In this study we modified Escherichia coli (E. coli) to autonomously display biotin on its cell surface via the engineered autotransporter antigen 43 (Ag43) and thus to bind to streptavidin modified surfaces. We could show that a biotin acceptor peptide (BAP) at the N-terminus of Ag43 is biotinylated in the cytoplasm, translocated to the cell surface and accessible to free or surface bound streptavidin. Flow cytometry measurements and fluorescence microscopy imaging of cells stained with fluorescently labelled streptavidin indicate that the biotinylation is strongly dependent on the intracellular levels of biotin and the biotin protein ligase BirA. Moreover, the staining pattern of Ag43 suggests that the majority of Ag43 is located at the cell poles. In addition, we modified Ag43 with the LOV2 domain of Arabidopsis thaliana, to control the accessibility of the displayed biotin through light controlled photocaging. To examine the effect of attachment on the fitness of E. coli, we used laser-assisted adsorption by photobleaching (LAPAP) to micro-pattern an abiotic surface with biotin. Such immobilized cells were able to grow for several generations and released their daughter cells into the medium. Aside from Ag43 alternative display mechanisms including OmpA (outer membrane protein A), INP (ice nucleating protein), AIDA-I (autotransporter) and FliC (flagellin), were investigated for biotin display, although only modified flagellin showed pronounced attachment to streptavidin. In a second part we used the Ag43 based biotin display system to fabricate bacteria-driven biohybrid microswimmers (bacteriabots). Bacteriabots combine synthetic cargo with motile bacteria that enable propulsion and steering. Although fabrication and potential use of such bacteriabots have attracted much attention, existing methods of fabrication require an extensive sample preparation that can drastically decrease the viability and motility of bacteria. Moreover, chemotactic behavior of bacteriabots in a liquid medium with chemical gradients has remained largely unclear. To overcome these shortcomings, we used our Ag43 based biotin display system to bind cells to streptavidin-coated cargo. We show that the cargo attachment to these bacteria is greatly enhanced by motility and occurs predominantly at the cell poles, which is greatly beneficial for the fabrication of motile bacteriabots. We further performed a systematic study to understand and optimize the ability of these bacteriabots to follow chemical gradients. We demonstrate that the chemotaxis of bacteriabots is primarily limited by the cargo-dependent reduction of swimming speed and show that the fabrication of bacteriabots using elongated E. coli cells can be used to overcome this limitation

MicroBioRobots for Single Cell Manipulation

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable micro actuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a bio-integrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 μm coated with a monolayer of the swarming Serratia marcescens. The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments, which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment

Micro- and nanotechnology for cell biophysics

Procedures and methodologies used in cell biophysics have been improved tremendously with the revolutionary advances witnessed in the micro- and nanotechnology in the last two decades. With the advent of microfluidics it became possible to reduce laboratory-sized equipment to the scale of a microscope slide allowing massive parallelization of measurements with extremely low sample volume at the cellular level. Optical micromanipulation has been used to measure forces or distances or to alter the behavior of biological systems from the level of DNA to organelles or entire organisms. Among the main advantages is its non-invasiveness, giving researchers an invisible micro-hand to “touch” or “feel” the system under study, its freely and very often quickly adjustable experimental parameters such as wavelength, optical power or intensity distribution. Atomic force microscopy (AFM) opened avenues for in vitro biological applications concerning with single molecule imaging, cellular mechanics or morphology. As it can operate in liquid environment and at human body temperature, it became the most reliable and accurate nanoforce-tool in the research of cell biophysics. In this paper we review how the above three techniques help increase our knowledge in biophysics at the cellular level

Actuation and control of microfabricated structures using flagellated bacteria

In this work methods of actuation and control of microfabricated structures are investigated using bacteria as configurable, scalable actuators. Bacteria offer many benefits as microfluidic actuators. They draw chemical energy directly from their environment, they can be operated in a wide range of temperature and pH, and literally billions of bacteria may be cultured within hours. Additionally, the well-documented responses of individual motile bacterial cells may be expected to scale up to arrays of cells. On this population scale, the cellular responses can be employed en masse creating controlled forces that actuate inorganic microfabricated elements. For these investigations the bacterium Serratia marcescens has been chosen. S. marcescens has properties that are particularly appropriate for engineering applications. When cultured on soft agar, the bacteria demonstrate a form of surface motility known as swarming. These investigations start with an experimental analysis of the swarming cell motility using a non-labeled cell tracking technique. The results of these studies reveal that the most energetic bacteria populate the progressing edge of the swarm. A technique of biocompatible microfabrication and chemical release of bacteria-driven microstructures is also presented. This method is used to pattern structure surfaces with the rigorous swarming cells by direct blotting. The self-coordinated motion of the cells is investigated for use as arrays of actuators. Control mechanisms are investigated to adjust rotational and translational motion using optical and electrical stimuli, respectively. The fundamentals of the electrokinetics are also investigated and integrated into a system demonstrating controlled manipulation of target objects and phenotypic chemical sensing.Ph.D., Mechanical Engineering -- Drexel University, 200

Light controlled motility of Escherichia coli. Characterization and applications

Characterization of wild type E. coli motility in response to light stimuli. Gene editing of bacteria to implement specifc functions (e.g. photokinesis). The engineered strain has been used to demonstrate that density modulation of photokinetic bacteria can be obtained by projecting spatially structured light on the sample. Additionally these bacteria have been also used as propelling units in microfabricated structures