Optimization of cell extract preparation procedure when using sonication for cell lysis.
(A) CFPS activity of extracts prepared from cells grown in 1 L of 2 × YTPG media and harvested at different optical densities. Extracts were prepared by sonicating 1.5 mL of cell suspension with 556 J. (B) CFPS activity of cell extracts prepared using varying ratios of wet cell pellet to buffer A for resuspension. Ratios are given in terms of wet cell weight in g per mL of buffer A. (C) CFPS activity of cell extracts prepared using varying centrifugation speeds for the first spin. The cell extracts were prepared without the run-off reaction and no second centrifugation. (D) The impact of run-off reaction times for preparation of active crude cell extracts. The run-off reaction was carried out at 37°C with shaking (250 rpm). In A and D, filled circles and open circles represent the CFPS activities of BL21 Star™ (DE3) and C495 cell extracts, respectively. In B and C, extracts were generated from BL21 Star™ (DE3). In B and C, black bars show the activity of a positive control cell extract prepared by 10 L fermentation for culture and impinge homogenization to lyse cells. sfGFP reported was based on the amount of active (fluorescent) sfGFP following a 4 hour CFPS reaction. Values represent averages and error bars represent standard deviation for at least 3 independent experiments.