We recently developed a continuous flow system to transform microorganisms in high throughput in a microfluidic device (Garcia et al., 2017). This system employs microfluidic channels that contain a bilateral constriction between the inlet and outlet electrode connections (length\u00a0= 3.0 mm, widthmin\u00a0= 50\u00a0\u03bcm, widthmax\u00a0= 2.0\u00a0mm, and height\u00a0=\u00a0100\u00a0\u03bcm). The constriction amplifies the electric field under an applied voltage between the inlet and outlet electrodes to levels sufficiently high to induce electroporation. During P. caudutus transformations, the cells were driven through the microfluidic device at flow rates of 50\u00a0\u03bcL\/min and 500\u00a0\u03bcL\/min, which correspond to residence times (i.e. pulse durations) of 20\u00a0ms and 2\u00a0ms, respectively.\u00a0Square wave pulses with, for example, 5 ms ON and 5 ms OFF cycles (50\u00a0% duty cycle) are applied to the microchannel through the dispensing needle. Therefore the cell viability cannot be accurately evaluated since only 50\u00a0% of the cells experience the electric field. The pulses are delivered from electrodes with alternating polarity between the pulses to reduce electrolytic effects at the electrode-buffer interface (Fig. 1). After flowing through the microchannel, each 200 \u03bcL cell sample is added to a 1.5 ml Eppendorf tube containing 1 ml of fresh growth media for cell recovery. The applied voltages we evaluated had amplitudes of 250\u00a0V (Emax\u00a0=\u00a01,500\u00a0V\/cm), 375\u00a0V (Emax\u00a0=\u00a02,250\u00a0V\/cm), and 500\u00a0V (Emax\u00a0=\u00a03,000\u00a0V\/cm) for each polarity. The non-uniform constriction in the microfluidic devices generates a variable electric field that is capable of transfecting cells while minimizing exposure to the highest electric field.\u00a0Figure 1: Electric field waveforms employed for transient and stable transfection of Bodo caudatus. Three independent electroporation systems were used for reproducible transfection including a) our microfluidic electroporation platform (Garcia et al., 2017), the NEPA21 square-wave transfection system (BulldogBio), and the MicroPulserTM exponential decay electroporator (Bio-Rad). Additionally, the b) signature waveforms for the NEPA21 square wave transfection system of \u2018poring\u2019 and \u2018transfer\u2019 pulses for electroporation are shown. Note: The time scale in Fig. 1a is a zoomed-in version of the red-dashed box from Fig. 1b.Garcia, P.A., Ge, Z., Kelley, L.E., Holcomb, S.J., and Buie, C.R. (2017) High efficiency hydrodynamic bacterial electrotransformation. Lab Chip\u00a0(17): 490-500.