The synthetic switch and sensors can be interfaced with the natural signaling pathways to control bacterial chemotaxis. (A) Without arabinose (top), the engineered strain produces CheW* (along with GFPmut3), making the strain respond to and attracted toward serine (serine chemotaxis). With arabinose addition (bottom), FimE inverts the invertible region, triggering expression of CheW (along with RFP). CheW preferably interacts with Tar, and the strain switches to aspartate chemotaxis. While P_BAD is an inducible promoter, P_trc and P_J23113 used in this paper are constitutive promoters. The dotted line is drawn to show the synthetic parts and devices (left) and the natural signaling pathways (right). CheB (methylesterase), CheR (methyltransferase), and CheZ (phosphatase) are peripheral components of the natural chemotaxis system and affect the concentration of phospho-CheY 27. (B) A map of the pChemoK plasmid is shown.

Assays for bacterial chemotaxis. (A) A gradient of aspartate or serine was created by spotting 10 μl of 10 mM attractant on semisolid agar (on the right sides of these images). Cultures were induced with 5 mM arabinose (bottom) or supplemented with 0.5% (w/v) glucose in order to repress potential leaky expression of P_BAD (top). Those shown are representative images of experiments that were performed at least three times. Red circles and green ovals were computationally generated as described in Figure 3B. (B) Image analysis was carried out using a Matlab® algorithm. (Top) Red circles indicate the positions where 10 μl of the culture was placed, and green ovals indicate the entire regions formed by bacterial chemotaxis. The regions surrounded by blue circles are assumed to be formed by random walks only. (Bottom) The area ratio is defined as the green oval area divided by the blue circle area. Bacteria executing random walks only would result in an area ratio of 1, whereas chemotactic bacteria attracted toward aspartate or serine would lead to a higher area ratio. Schematic images are shown for four area ratio values to serve as a guide. (C) Quantitative analysis of bacterial chemotaxis. Data are the averages and standard deviations of three images taken from three experiments performed on three different days. No attractant spotted (−); 10 μl of 10 mM aspartate spotted (+ Asp); 10 μl of 10 mM serine spotted (+ Ser). Uninduced (− Ara) and induced (+ Ara).

Characterization of the fim switch using two-color flow cytometry. (A) GFPmut3 (green) and RFP (red) were placed on either side of the fim invertible region. (B–F) FimE-promoted switching occurred more quickly in LB medium than minAaa medium. ▲ = the average RFP fluorescence (normalized); ■ = the average GFPmut3 fluorescence (normalized). Normalization was done by the following formula: [log(F) − log(F_B)]/[log(F_M) − log(F_B)] where F, F_B, and F_M are sample, background, and maximum fluorescence levels, respectively. The background fluorescence values were obtained from control cells lacking the fluorescent protein genes (CAV8). Data are the averages and standard deviations of three replicates. (B) Cultures in LB medium with 6 hr induction. (C) Cultures in LB medium containing 5 mM arabinose (see Figure S2 for the OD₆₀₀ data). (D) Two-color flow cytometry data are shown for cultures in LB medium. (Top) Cultures were induced for 6 hr with 0 mM (left), 0.02 mM (middle), and 5 mM (right) of arabinose. (Bottom) Cultures were induced with 5 mM arabinose for 0 hr (left), 4 hr (middle), and 8 hr (right). The dotted lines are drawn as a guide to the eye, separating the green (lower right), red (upper left), and green/red (upper right) regions. (E) Cultures in minAaa medium with 16 hr induction. (F) Cultures in minAaa medium containing 5 mM arabinose (see Figure S2 for the OD₆₀₀ data).

Analysis of genetic program with various genes knocked out. The four deletion mutants lack one of the four genetic parts required for the orthogonal chemotaxis signaling, and chemotaxis assays using these mutants were carried out as described in Figure 3. Data are the averages and standard deviations of three images taken from three experiments performed on three different days (for representative images, see Supplementary Information). The black lines, indicating the average values from Figure 3C, are drawn to serve as a comparison guide. No attractant spotted (−); 10 μl of 10 mM aspartate spotted (+ Asp); 10 μl of 10 mM serine spotted (+ Ser). Uninduced (− Ara) and induced (+ Ara). (A) Chemotaxis assay using the cheW deletion mutant. The remaining CheW* after fim switching may result in the higher area ratio than expected (+ Ser + Ara). (B) Chemotaxis assay using the tar deletion mutant. Crosstalk between Tsr* and CheW seems to increase significantly when the cognate partner Tar does not exist (+ Ser + Ara). (C) Chemotaxis assay using the cheW* deletion mutant. Crosstalk between Tsr* and CheW was confirmed in the absence of cheW* (+ Ser + Ara). (D) Chemotaxis assay using the tsr* deletion mutant. No crosstalk was observed between Tar and CheW* (+ Asp − Ara).