Introduction
Cell free protein synthesis (CFPS) is a method of producing proteinsin vitro , which is widely used to manufacture synthetic biology
sensors (Pardee et al., 2016; Salehi et al., 2017; Takahashi et al.,
2018), prototype proteins (Dopp & Reuel, 2020; Dopp, Rothstein,
Mansell, & Reuel, 2019), discover metabolic pathways (Dudley, Nash, &
Jewett, 2019; Jaroentomeechai et al., 2018), and to manufacture some
protein therapies (Cai et al., 2015; Yin et al., 2012; Zawada et al.,
2011). In a typical reaction, the protein is expressed by combining cell
extract with energy supplements and a DNA template. Bacterial cells,
most commonly a productive E. coli strain with protease knockouts
(BL21 DE3 star), are lysed to produce the cell extract which contains
required intercellular machinery for the reaction, like ribosomes and
metabolic enzymes (Smith, Wilding, Hunt, Bennett, & Bundy, 2014). As an
active cell extract is key for optimal CFPS, many groups have focused on
optimizing and streamlining the extract production procedure (Kwon &
Jewett, 2015; Shrestha, Holland, & Bundy, 2012). Sonication is a
convenient and cost-effective method for lysing small cell batches
(<20 g wet cell weight), as opposed to use of more
capital-intensive homogenizer or French press at larger scale (Dopp &
Reuel, 2018; Kwon & Jewett, 2015; Shrestha et al., 2012).
Sonication uses oscillating ultrasonic waves to lyse the outer membranes
of cells via cavitation. A probe transducer (Harrison, 1991; Islam,
Aryasomayajula, & Selvaganapathy, 2017) is inserted into the fluid
vessel (Fig 1a) and its tip oscillates causing rapid back and forth
fluid motion, leading to pressure waves that create and compress bubbles
(Jamshidi, 2013). The resulting shock waves from collapsing bubbles are
sufficient to rupture cell membranes. The local temperature, in a very
small region of cavitation, increases by thousands of degrees K (Wang,
Yuan, & Hale, 2016), but quickly dissipates. Over time this causes
buildup of thermal energy in the sonication tube causing temperature
rise (Islam et al., 2017; Wang et al., 2016). Additionally, sonication
induces fluid flow via ultrasonic waves in a process called acoustic
streaming (Nyborg, 1953). Proper mixing in the cell suspension vessel is
necessary for efficient sonication, to eliminate local hot spots (Zhou
et al., 2010); otherwise a small fraction of suspension will be over
sonicated (causing unwanted temperature rise) and the bulk of the
suspension will sit idle. For cells collected from typical shake flask
growths (~1-20 g wet cells, prepared at 1 g/ml buffer),
sonication is typically performed in snap-cap microcentrifuge 1.5 mL
tubes, but can also be done in 5, 15, and 50 mL conical vessels (Fig
1b). Most protocols carefully specify tip height placement or suggest
manual motion of the tip to ensure the suspension is mixed and subject
to the same level of ultrasonic exposure. The production of viable cell
lysate is proportional to cavitation and the extent of cavitation is
dependent on the power input (Harrison, 1991; Sutkar & Gogate, 2009;
Wang et al., 2016). Higher power density results in faster cell lysis,
but greater heat accumulation. Researchers concluded that the total
sonication energy input from the tip ultimately converts to thermal
energy very near to the tip (Chivate & Pandit, 1995; Prabhu, Gogate, &
Pandit, 2004). It is documented that extract conditioning temperature
rise affects CFPS yield (Kigawa et al., 2004), which agrees with our
biophysical understanding of denaturing and loss of function at elevated
temperatures, typically > 42C (de Groot & Ventura,
2006; Farewell & Neidhardt, 1998). It is also observed that prolonged
ultrasound exposure can have adverse effects on biological molecules,
namely the formation of hydrogen, hydroxide and peroxide radicals and
degradation of protein and enzymes (Islam et al., 2017; Miller, Miller,
& Brayman, 1996; Rokhina, Lens, & Virkutyte, 2009; Save, Pandit, &
Joshi, 1997).