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).