Communicated by Howard C. Berg, Harvard University, Cambridge, MA (received for review November 19, 2002)
Device for microfluidic chemotaxis assays. The main channel is 18 mm long at the edge and 21 mm long in the center. Its height is 8 μm. The bacterial inlet is 151 μm wide, each buffer inlet is 1.51 mm wide, and the total width of the channel is 3.18 mm. The width of each outlet port at its junction with the main channel is 200 μm. The vertical arrow indicates the direction of increasing chemoeffector concentration across the channel.
Distribution of cells in the absence of gradients. Cells were grown as described. Cells of motile strains swam vigorously at the time of harvesting. (A) Washed cells in chemotaxis buffer were introduced into the microfluidic device and counted as described. The number of cells counted in each outlet port is given. The total number of cells was normalized to the mean number (2,105) of cells counted during seven runs with the WT strain. WT cells (●), aspartate-blind cells (▵), receptorless cells (□), flagellated, nonmotile cells (◊), and nonflagellated cells (○). The horizontal line with short dashes shows the no-cell baseline, and the vertical dotted line marks the middle of the device, corresponding to outlet 11.5. (B) The distribution of WT cells (from A), with error bars. In seven trials the standard error in the counts for well populated channels was <10%. The standard error across all outlets was ≈20%. The line with long dashes represents the best-fit Gaussian curve (σ = 3.6) for WT cells, and the line with short dashes represents the curve calculated for the case of diffusion from an extended source of limited extent (20). Note that there is little difference between the latter and the normal curve.
Responses of WT cells to attractant and repellent. (A) Normalized cell counts were determined as in Fig. 2 for: 3.2 nM l-aspartate (▵), 10 nM l-aspartate (□), and 1 mM l-aspartate (◊). (B) Normalized cell counts for: 10 μM Ni2+ (▵), 1 mM Ni2+ (□), and 10 mM Ni2+ (◊). The curve with long dashes shows the normal distribution in the presence of buffer (Fig. 2B). The vertical line with short dashes marks the middle of the device.
Differential response of WT cells to attractant and repellent. Difference calculations are described in the text. (A) Difference in normalized cell counts in the presence of l-aspartate: buffer only (●), 3.2 nM (▵), 10 nM (□), and 1 mM (◊). (B) Difference in normalized cell count for WT cells in the presence of Ni2+: 10 μM (▵), 1 mM (□), and 10 mM (◊). (C) Difference in normalized cell count for WT cells in the presence of l-serine: 1 μM (▵), 1 mM (□), and 10 mM (◊). (D) Difference in normalized cell count in the presence of l-leucine: 1 μM (▵), 100 μM (□), and 10 mM (◊).
CPC and CMC values for WT and mutant cells. WT cells (▴), serine-blind cells (■), and aspartate-blind cells (⧫). (A) CPC with l-leucine. (B) CMC with l-leucine. (C) CMC with l-aspartate. (D) CMC with Ni2+. (E) CMC with l-serine.
| Genotype | Reference | |
|---|---|---|
| Strains | ||
| RP437 | thr(Am)1, leuB6, his-4, metF(Am)159, eda-50, rpsL1356, thi-1, ara-14, mtl-1, xyl-5, tonA31, tsx-78, lacY1, F− | 7 |
| MM509 | RP437 eda+ Δ(tar-tap)5201 | 8 |
| SW10 | RP437 thr+ Δtsr7021 | This study |
| VB13 | RP437 eda+ thr+ Δtsr7021 Δ(tar-tap)5201 trg∷Tn10 | 9 |
| MM5000 | Rp437 eda+ ΔmotAB | 10 |
| RP3098 | RP437 Δ(flhA-flhD) | 11 |
| Plasmids | ||
| pBAD18 | araC paraBAD bla (Ampr) | 12 |
| pBJC100 | pBAD18 with gfp cloned behind paraBAD | 13 |
