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Abstract

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.