Fig. 2. Extensions to the TIS method.

a | Physical separation of mutant populations based on motility. This includes assaying genes for motility by inoculating transposon-insertion sequencing (TIS) mutants on an agar plate (yellow circle and beige spot) and separating the inner mutant pool (less motile) from the outer mutant pool (more motile). b | In density–TraDISort, mutant populations can be separated into top, middle and bottom fractions (shown by horizontal orange bands) on the basis of their increased or decreased cellular density using a Percoll gradient and centrifugation. c | Separation of single mutants using fluorescence (TraDISort). The mutant pool is treated with the fluorescent marker ethidium bromide (EthBr) and subjected to fluorescence-activated cell sorting, where each cell is sorted with use of a laser (horizontal red line) on the basis of its fluorescence (shown as green), reporting on efflux activity. d | Encapsulation, growth and sorting with microfluidics of single mutants in droplets for droplet transposon sequencing (dTn-Seq). Each single mutant with different growth rates (in the schematic on the left, low, medium and high levels of growth are represented as blue, orange and green background colours, respectively) will grow independently within its own droplet (grey circle), eliminating the effects of interactions between mutants. A final sorting step, based on cell fluorescence or microscopy, can also be added. Alternatively, cell-containing droplets (blue droplet on the right) can undergo multiple layers of re-encapsulation, so that an encapsulated mutant can be encapsulated within another droplet containing a different cell (shown in yellow; this can be another mutant, another bacterial cell or a host cell) and signals can freely diffuse between the layers (shown as red bolts) to allow cell interactions to be investigated by sorting those cell combinations that have altered fitness and grow at different rates, or those that can be separated by sorting based on markers, such as alterations of cell morphology observed with a microscope.