CFPS extract was prepared by sonication, as previously reported (47). Briefly, E. coli BL21 Star (DE3) (Thermo Fisher Scientific) was grown in 2× YTPG media at 37°C. T7 polymerase expression was induced at an OD600 (optical density at 600 nm) of 0.6 to 0.8 with 1 mM isopropyl-β-d-1-thiogalactopyranoside. Cells were grown at 30°C to a final OD600 of 3.0, at which point cells were pelleted by centrifugation at 5000g for 15 min at 4°C. Cell pellets were then washed three times with cold S30 buffer [10 mM tris-acetate (pH 8.2), 14 mM magnesium acetate, and 60 mM potassium acetate] and pelleted at 5000g for 10 min at 4°C. After the final wash, cells were pelleted at 7000g for 10 min at 4°C, weighed, flash-frozen in liquid nitrogen, and stored at −80°C. For lysis, cell pellets were suspended in 1 ml of S30 buffer per 1 g of wet cell mass, and cells were transferred into 1.5-ml microcentrifuge tubes and placed in an ice-water bath to minimize heat damage during sonication. The cells were lysed using a Q125 Sonicator (Qsonica) with a 3.175-mm-diameter probe at 20 kHz and 50% amplitude. The input energy was monitored, with 640 J used to lyse 1 ml of suspended cells. The lysate was then centrifuged once at 12,000g at 4°C for 10 min. Cell extract was aliquoted, flash-frozen on liquid nitrogen, and stored at −80°C. Alternatively, for classroom settings where it is not practical to generate or obtain FD-CF reactions, similar cell-free systems are available commercially from companies such as Promega (L1130).

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