Once the optimal IPTG concentration GSK-3 assay was obtained, additional cell-based assays were performed against a panel of known antibiotics of different chemical classes to obtain fold sensitization values [the ratio between the IC50 value (the concentration at which cell growth inhibited 50% compared with control) under noninduced condition and that of induced condition]. Based on the result of a time-course Sau3AI digestion (Fig. S1a, Supporting Information), the optimal partial digestion time was 4 h to generate DNA fragments of

appropriate size for library construction. Ligation mixtures were transformed into E. coli DH5α competent cells. Insert cloning efficiency analysis (Fig. S1b) indicated that the cloning efficiency for this library was 92%. To increase the randomness of the genomic DNA generated,

an alternative genomic library was constructed using a blunt-end producing restriction endonuclease (CviKI-1). The cloning efficiency of the CviKI-1-based library (termed Library C) was 90%. To screen for inducible growth inhibitory recombinant clones, transformants MAPK Inhibitor Library were grown overnight with chloramphenicol in the presence or absence of inducing IPTG. An example of screening plates and sensitive clones is shown (Fig. S1c). A total of 1500 confirmed IPTG-sensitive clones were mafosfamide obtained from screening 250 000 individual transformants. Only 675 of the 1500 confirmed clones were sequenced. An example of inducer-dependent inhibition of growth of asRNA clone PT113 is shown in Fig. S1d. Plasmid DNAs from a total of 675 confirmed inducer sensitive clones were sequenced. It was determined that enough clones were analyzed because more analysis leads to identification of duplicates, suggesting that the

phenotypic screening process under the condition scheme is approaching saturation. Among the sequenced clones, 134 separate clones contained insert DNA sequences derived from and in antisense orientation to known essential genes based on PEC database (Table 1). For most of the essential genes targeted by asRNAs, multiple gene silencing asRNA constructs were discovered, with rplF gene (encoding 50S ribosomal subunit protein L6) being ‘hit’ the most (17 times) (Table 1). Because many essential genes engaging in a cellular process are usually clustered in an operon, many essential operons are targeted by a multitude of asRNAs, especially the operons for ribosomal protein genes. For example, rplN operon that contains 11 essential genes was ‘hit’ by 17 unique asRNAs (Fig. 1a, with two asRNAs not shown owing to space limit). On an individual gene level, four unique asRNAs were found to target fusA gene (Fig. 1b), while another four to target rpoC gene (Fig. 1d).