Figure 6: Maximum temperature (y-axis) estimated for different power density (x-axis) and pulse time (5-10,10-10,15-10 and 20-10 on-off pulses respectively) for each of the four tubes – a) 1.5 mL microcentrifuge tube b) 5 mL microcentrifuge tube c) 15 mL conical tube d) 50mL conical tube.
To provide clarity on use of these master plots we provide an example. Assume a cell growth yields 3 g of cells, to make 3 mL of cell suspension for sonication and the specific strain threshold temperature is 320K (empirically observed). First a sonication tube with adequate volume is selected – in this case the 1.5 mL tube is too small, and the 5 mL tube is a suitable choice. Next, draw a horizontal line at the max temperature (in this case 320K) for the selected tube (dotted line in Fig 6b) which will intersect lines for four different pulse condition (5-10,10-10,15-10 and 20-10 on off pulses). The power density for these pulse conditions are then read from the x-axis at these points (4.5, 3, 2.5, and 2.2 W/mL respectively). Then the sample volume (in mL) is multiplied with the power density for the selected pulse condition to get the maximum permissible power. In the case of 3 mL samples in the 5 mL tube at the 5-10, 10-10, 15-10 and 20-10 s on/off pulse settings, the max power settings obtained are 13.5, 9, 7.5, 6.6 W respectively. Now one can tune the power setting with reference to the sonicator calibration chart (provided by vendor or empirically determined, see Supplement Fig 6,7) to select an input power that will not exceed the threshold temperature. In the case of this example, if less than 6.6 W power is used with 20-10 s on/off pulses the maximum temperature will always be below 320K regardless of time sonicated. The next step would be to process a few batches of cells at these conditions to determine the appropriate energy density (J/mL) and thus overall length of sonication (number of pulse) cycles on yield. Using the optimal energy density of 550 J/mL from the BL21 DE3 star cells, the sonication duration would be 570 seconds or 19 cycles.