Additive manufacturing enables the generation of 3D structures with predefined shapes from a wide range of printable materials. However, most of the materials employed so far are static and do not provide any intrinsic programmability or pattern‐forming capability. Here, a low‐cost 3D bioprinting approach is developed, which is based on a commercially available extrusion printer that utilizes a DNA‐functionalized bioink, which allows to combine concepts developed in dynamic DNA nanotechnology with additive patterning techniques. Hybridization between diffusing DNA signal strands and immobilized anchor strands can be used to tune diffusion properties of the signals, or to localize DNA strands within the gel in a sequence‐programmable manner. Furthermore, strand displacement mechanisms can be used to direct simple pattern formation processes and to control the availability of DNA sequences at specific locations. To support printing of DNA‐functionalized gel voxels at arbitrary positions, an open source python script that generates machine‐readable code (GCODE) from simple vector graphics input is developed.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far, only a few viable approaches are available for the printing of gels hosting live Escherichia coli bacteria. Here, we develop a gentle extrusionbased bioprinting method based on an inexpensive alginate/agarose ink mixture that enables printing of E. coli into three-dimensional hydrogel structures up to 10 mm in height. We first characterize the rheological properties of the gel ink and then study the growth of the bacteria inside printed structures. We show that the maturation of fluorescent proteins deep within the printed structures can be facilitated by the addition of a calcium peroxide-based oxygen generation system. We then utilize the bioprinter to control different types of interactions between bacteria that depend on their spatial position. We next show quorum-sensing-based chemical communication between the engineered sender and receiver bacteria placed at different positions inside the bioprinted structure and finally demonstrate the fabrication of barrier structures defined by nonmotile bacteria that can guide the movement of chemotactic bacteria inside a gel. We anticipate that a combination of 3D bioprinting and synthetic biological approaches will lead to the development of living biomaterials containing engineered bacteria as dynamic functional units.

Pattern formation processes play a decisive role during embryogenesis and involve the precise spatial and temporal orchestration of intricate gene regulatory processes. Synthetic gene circuits modeled after their biological counterparts can be used to investigate such processes under well-controlled conditions and may, in the future, be utilized for autonomous position determination in synthetic biological materials. Here, we investigated a three-node feed-forward gene regulatory circuit in vitro that generates three distinct fluorescent outputs in response to varying concentrations of a single externally supplied morphogen. The circuit acts as a band detector for the morphogen concentration and, in a spatial context, could serve as a stripe-forming gene circuit. We simulated the behavior of the genetic circuit in the presence of a morphogen gradient using a system of ordinary differential equations and determined optimal parameters for stripe-pattern formation using an evolutionary algorithm. To analyze the subcircuits of the system, we conducted cell-free characterization experiments and finally tested the whole genetic circuit in nanoliter-scale microfluidic flow reactors that provided a continuous supply of cell extract and metabolites and allowed removal of degradation products. To make use of the widely employed promoters PLlacO‑1 and PLtetO‑1 in our design, we removed LacI from our bacterial cell extract by genome engineering Escherichia coli using a pORTMAGE workflow. Our results show that the band-detector works as designed when operated out of equilibrium within the flow reactors. On the other hand, subcircuits of the system and the whole circuit fail to generate the desired gene expression response when operated in a closed reactor. Our work thus underlines the importance of out-of-equilibrium operation of complex gene circuits, which cannot settle to a steady-state expression pattern within the finite lifetime of a cell-free expression system.

Bioprinting of engineered bacteria is of great interest for applications of synthetic biology in the context of living biomaterials, but so far only few viable approaches are available for the printing of gels hosting live Escherichia coli bacteria. Here we develop a gentle bioprinting method based on an alginate/agarose bioink that enables precise printing of E.coli into three-dimensional hydrogel structures up to 10 mm in height. Addition of a calcium peroxide-based oxygen generation system enables maturation of fluorescent proteins deep within the printed structures. We utilize spatial patterning with the bioprinter to control different types of chemical interaction between bacteria. We first show quorum sensing-based chemical communication between engineered sender and receiver bacteria placed at different positions inside the bioprint, and then demonstrate the fabrication of barrier structures defined by non-motile bacteria that can guide the movement of chemotactic bacteria inside a gel.