Figure 1.

High-throughput screen to identify small molecule inhibitors of prokaryotic protein synthesis. (a) Overview of the economical, high-throughput bacterial lysate-based cell-free protein synthesis reaction. Shown here is a circular DNA template containing luciferase downstream of a T7 promoter (pT7). T7 RNA polymerase was spiked into bacterial lysate, and the reaction was incubated with test compounds for 1.5 hours before the addition of luciferin for measurement of the luciferase signal. (b) Map of plasmid pIVEX2.3d-luc used for a luciferase template. (c) Dose-dependent sensitivity of the protein synthesis inhibition reaction when incubated with varying concentrations of kanamycin. 1.2 uM of kanamycin was found to sufficiently inhibit the reaction and was used to terminate all subsequent reactions at 1.5 hours. Error bars represent the standard deviation for each condition. Abbreviations: Prom - Promoter, Term - Terminator, RBS - Ribosome binding sequence (AGGAGA), Stop - Tandem stop codons (TAATAA), Ori - Origin, Amp - Ampicillin, R - Resistance