External tuning of the position and bandwidth of the band-pass filter. (A) Relation between IPTG and the MIC_Amp for strains RH06 and RH09 in liquid LB at 37 °C in a 96-deep-well format. (B) Induction of the ampC promoter by Amp. Total culture fluorescence (i.e., induction of GFP) was measured as a function of Amp for RH05 cells in liquid LB at 37 °C in a 96-deep-well format. Fluorescence (arbitrary units) was normalized by the culture's OD₆₀₀. The values are the mean of the ratio (n = 3), and the error bars reflect the SD. The value for cells in the absence of Amp was 120 ± 20. The value is plotted as 0 for Amp ≥1 μg/mL because the cells did not grow. (C) The growth of cells in liquid LB containing 20 μg/mL Tet was detected by measuring the OD₆₀₀ of the liquid culture and is presented in the form of a heat map as a function of Amp concentration (y axis) and cellular β-lactamase activity (x axis). The level of cellular β-lactamase is controlled by the concentration of IPTG (see A) and expressed as MIC_Amp. Strain RH09 was used to observe the behavior of cells with very low levels of β-lactamase activity. (D) Selected data from the heat map illustrate how cellular β-lactamase activity (controlled by IPTG and quantified as MIC_Amp) can be used to tune the position of the band-pass filter for Amp. (E) Selected data from the heat map illustrate how Amp can be used to tune the position of the band-pass filter for β-lactamase activity. Values in C–E are the mean (n = 3), and error bars reflect the SD. (F) The bandwidth can be externally tuned by the concentration of Tet. The growth of RH06 cells in liquid media containing IPTG (300 μM), different amounts of Tet (different lines), and Amp (x axis) was detected by measuring the OD₆₀₀ of the liquid culture. The concentrations of Tet were 0, 5, 6, 7, 8, 9, 10, 15, and 20 μg/mL. The OD₆₀₀ is normalized to the maximum value for each Tet concentration tested. The detailed conditions for C–E can be found in Table S3.

A synthetic model system for morphogen-induced pattern formation in development. RH01 cells were plated with an Amp disc in the center (conditions for plating as in Fig. 3A). An Amp gradient elicits a growth pattern that fits the French flag model (10) and consists of 3 different cell types with a defined spatial relation with each other and the Amp source.

Design of the bacterial band-pass filter. (A) Schematic depiction of the essential components of the band-pass filter. Plasmids pTS1 and pDIM-C8 are compatible. In the absence of sufficient cellular β-lactamase (BLA) activity for hydrolysis of Amp, cell wall synthesis is compromised and cells cannot proliferate. In addition, cell wall breakdown results in the accumulation of aM-pentapeptide (aM-Pp), which induces the ampC promoter via interactions with AmpR (6, 7), resulting in the production of TetC (which confers Tet resistance) and GFP. However, the level of Amp necessary to induce ampC is lower than the level that prevents the growth of E. coli cells (7). BLA gene expression is regulated through IPTG induction of the tac promoter, which is repressed by LacI. Complete plasmid maps are provided in Fig. S1. (B) Conceptualization of the genetic circuit as an incoherent type 3 feed-forward loop (4). In order for growth to occur in the presence of Tet, the Amp concentration must not be above the MIC_Amp but TetC must be present as a result of induction by Amp (K_{ind, Amp}). The values of MIC_Amp and K_{ind, Amp} are approximate and for when BLA is absent. BLA serves to attenuate the Amp signal entering the feed-forward loop. (C) Schematic depiction of how the combination of positive and negative genetic selection results in band-pass behavior. Band-pass behavior results when the high-pass cutoff (f₁) is set at a lower enzyme activity than the low-pass cutoff (f₂).

Bacterial pattern formation using the band-pass filter circuit. Liquid cultures were spread evenly on 86-mm-diameter round or 91-mm square LB-agar plates containing Tet and/or IPTG. Ninety minutes after plating, filter paper discs were placed in the center or at the indicated locations and were spotted with Amp, IPTG, or Tet. Plates were incubated at 37 °C. Visible light and fluorescent images are shown. The detailed conditions for all plates can be found in Table S4 and Fig. S5. (A) Cells on IPTG plates with different concentrations of Tet and an Amp disc in the center. (B) Cells on Tet/IPTG plates with Amp discs of different concentrations in the center. (C) Cells on Tet plates with different concentrations of IPTG and an Amp disc in the center. (D) A 1:1:1 mixture of RH01, RH05, and RH06 cells (strains with different levels of β-lactamase activity) on Tet/IPTG plates with an Amp disc in the center. (E) Overlapping radial concentration gradients of 2 compounds. The black line comprising all points equidistant from the 2 point sources marks the location where the ratio of the 2 compounds' concentrations is a constant. (F) Cells on Tet plates with discs of Amp, IPTG, or Tet at the indicated positions.