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.