Engineering robust and tunable spatial structures with synthetic gene circuits

Abstract

Controllable spatial patterning is a major goal for the engineering of biological systems. Recently, synthetic gene circuits have become promising tools to achieve the goal; however, they need to possess both functional robustness and tunability in order to facilitate future applications. Here we show that, by harnessing the dual signaling and antibiotic features of nisin, simple synthetic circuits can direct Lactococcus lactis populations to form programmed spatial band-pass structures that do not require fine-tuning and are robust against environmental and cellular context perturbations. Although robust, the patterns are highly tunable, with their band widths specified by the external nisin gradient and cellular nisin immunity. Additionally, the circuits can direct cells to consistently generate designed patterns, even when the gradient is driven by structured nisin-producing bacteria and the patterning cells are composed of multiple species. A mathematical model successfully reproduces all of the observed patterns. Furthermore, the circuits allow us to establish predictable structures of synthetic communities and controllable arrays of cellular stripes and spots in space. This study offers new synthetic biology tools to program spatial structures. It also demonstrates that a deep mining of natural functionalities of living systems is a valuable route to build circuit robustness and tunability.

Figure 1.

A simple gene circuit that produces robust spatial band-pass patterns. (A) Circuit design. The circuit consists of a two-component nisin signaling system (nisRK) and a nisin inducible green fluorescence reporter (gfp) only. (B) Differential responses of the circuit to external nisin. A low level of nisin concentration fails to trigger fluorescence production or cell death, resulting in cells viable but non-glowing. A medium level of nisin concentration induces the expression of green fluorescence gene without killing the cells, enabling the cells to glow. A high level of nisin causes cell death, resulting in a failure in producing fluorescence. (C) Schematic of the band-pass behavior in space. (D) Confirmation of the signaling and antibacterial features of nisin in liquid settings (n ≥ 3). (E) Spatial band-pass pattern emerged from a cellular lawn loaded with the circuit. A green fluorescence stripe forms around a nisin-emitting well (orange well). Between the stripe and the well is the inhibition zone caused by the antibacterial feature of nisin. Scale bar: 5 mm. (F) Single-cell images of the band-pass populations across space. Top row: brightfield images; bottom row: fluorescence images. Scale bar: 10 μm. (G) Experimental setup for remote pattern printing on a solid agar plate. The cellular lawn growing at the bottom of the plate is covered by a solid agar layer; a mask soaked with nisin is placed on top of the agar. (H) Demonstration of pattern printing. The pattern of the mask (i love (heart) GAMES) was remotely printed to the cellular lawn at the bottom through nisin diffusion, producing a glowing structure that reflect the edge of the mask pattern. Scale bar: 2 cm. (I) Various traffic sign patterns created using the procedure described in panel G. Scale bar: 5 cm.

Figure 2.

Pattern fine-tuning with cellular nisin immunity and external nisin concentration. (A) Circuit design. A nisin immunity gene (nisI) is introduced into the original circuit in Figure 1A to alter cellular sensitivity to nisin. (B) Schematic illustration of enhanced nisin immunity conferred by the expression of the nisin immunity gene. (C) Colony forming unit counting as a function of external nisin for cells containing different levels of nisin immunity. Data are presented as mean ± SD (n ≥ 3). (D) Spatial band-pass patterns emerged from populations with or without nisin immunity. For the cellular lawns containing the nisin immunity gene (bottom row), the inner diameters, defined by cellular resistance to nisin, are much smaller than those of the cells lacking nisin immunity (top row). Here, the orange circles are wells loaded with nisin. Scale bar: 5 mm.

Figure 3.

Various spatial patterns directed by nisin-producing bacteria. (A–C) Experimentally observed patterns of cellular populations directed by nisin producing bacteria. (A) Patterns of single-species responder populations. (B) Patterns of mixed, two-species responder populations. (C) Controls. Each experiment was repeated at least three times. Representative images from experiments are shown. The details of the responder and producer cells are available in Table 1. (D–F) Patterns emerged from the simulations of a mathematical model. The settings are identical to those of the experiments (A–C).

Figure 4.

Autonomous emergence of distinct but predictable spatial structures from mixed bacterial populations. (A) Spatial patterns of bacterial consortia from the predictions of our computational model. Qualitatively distinct patterns develop as the species composition and initial relative abundance are varied. (B) Experimental validation of spatial structures of bacterial populations that have the same species composition and relative abundance with those of the computational counterparts in panel A. Scale bar: 0.3 mm.

Figure 5.

Controllable spatial arrays of stripes and spots programmed with the engineered producer (PiG) and responder (iRS) cells. (A). Schematic for the initial setup of stripe arrays. (B–E) Differential patterns of red fluorescence stripes (responder cells) develop with the increase of the spacing between the green fluorescence strips (producer cells). Scale bar: 4 mm. (F) Schematic for the initial setup of spot arrays. (G–K). Distinct patterns of red spots (responder cells) form with the alteration of the green spots (producer cells). Scale bar: 4 mm. Each experiment was repeated at least three times. Representative images from experiments are shown.