e102 Nucleic Acids Research, 2008, Vol. 36, No. 16 PAGE 8 OF 10 Figure 3. Identiﬁcation of target genes associated with thermotolerance. (A) Analysis of thermotolerance by overexpression or knockout of the cpxP and/or ompW genes and knockout ( ) of the marRAB operon. Growth of cells was monitored after incubation for 2 h at 378C (No shock) or for 2 h at 558C (Heat shock). The triangles above each panel indicate tenfold serial dilutions (1 : 1 to 1 : 1000, left to right) of spotted cells. (B) Growth proﬁles in LB medium (100 ml) at 37 and 508C, respectively. Inset graph shows growth proﬁles of LT2 cells expressing T2 ATF on low-copy plasmid in LB medium (100 ml) at 508C. The O.D.s were measured with a spectrophotometer at 600 nm at diﬀerent time intervals. The growth experiments were performed in triplicate. Error bars represent SD. C, the wild-type E. coli transformed with a control plasmid without any previous selection. cpxP and ompW, cells overexpressing cpxP and ompW, respectively. cpxP + ompW, cells co-expressing cpxP and ompW. cpxP, cpxP knockout mutant. ompW, ompW knockout mutant. marRAB, marRAB knockout mutant. DISCUSSION The intricate interaction of multiple genes throughout the entire gene expression network controls the phenotypic var- iations in cells and organisms. Herein, we have demon- strated the applicability of ATF libraries to induce genetic perturbations (through transcriptional activation or repres- sion) and thus to induce phenotypic variations in E. coli. Transcription factors have modular structures that con- sist of a DNA-binding domain and an eﬀector (activation or repression) domain. Libraries of proteins that contain only a DNA-binding domain have been used to repress transcription (12,17). However, DNA-binding domains that do not contain an eﬀector domain cannot activate the expression of genes, because activation of transcrip- tion depends upon the activity of an eﬀector domain. Using CRP as an eﬀector domain, our ATFs can activate the expression of genes. Furthermore, prokaryotic tran- scription factors can have dual activity as either an acti- vator or a repressor (36). Activation or repression mediated by transcription factors is linked to the distance of the regulator-binding sites relative to promoters (36). The preferred sites for activators are located between