e102 Nucleic Acids Research, 2008, Vol. 36, No. 16 To construct an ATF library, a DNA segment that encoded CRP D1 was PCR-ampliﬁed from MG1655 genomic DNA using the following forward (CRP-F) and reverse (CRP-R) primers, respectively: CRP-F, 50-CATG CCATGGTGCTTGGCAAACCGCAAACA-30 [which contained an NcoI site (bold)]; and CRP-R, 50-CCG GAATTCACCGGTAGAACAGCCGACAATCTGAC-30 [which contained an AgeI site and an EcoRI site (bold)]. The PCR-ampliﬁed CRP D1 DNA, after digestion with NcoI and EcoRI, was cloned into pETtac that had been digested with the same enzymes, producing pETtac- CRP D1. Equal amounts of each single-ﬁnger plasmid were mixed together to form a pool. The single-ﬁnger plasmid pool was digested with XmaI and EcoRI and then ligated into the AgeI/EcoRI-digested pETtac- CRP D1 to generate a series of pETtac-CRP D1-1ZF library vectors, each encoding CRP D1 and one ZF. After transformation of E. coli XL1-Blue with these plasmids, the pETtac-CRP D1-1ZF library vectors were puriﬁed from the transformants. Subsequently, the pETtac-CRP D1-1ZF library vectors that encoded CRP D1 and one ZF were digested with AgeI and EcoRI and then ligated to the pool of the XmaI/EcoRI-digested single-ﬁnger. After transfor- mation of E. coli with the products of these ligation reac- tions, plasmids were puriﬁed, which now encoded CRP D1 and two ZFs (the pETtac-CRP D1-2ZF library vectors). To the AgeI/EcoRI-digested pETtac-CRP D1-2ZF library vectors, the pool of XmaI/EcoRI-digested single- ﬁnger was ligated to generate the ﬁnal ATF library used in the experiments herein, which encoded CRP D1 and three ZFs (the pETtac-CRP D1-3ZF library vectors) (14). Screening for desirable phenotypes E. coli MG1655 was electrotransformed with 10 ng of ATF library vector described above, with a transforma- tion eﬃciency of 1 108 cfu/mg DNA. The resulting 106 transformants which exceeded the complexities of the ATF libraries were analyzed for thermotolerance, osmo- tolerance and cold-tolerance upon ATF expression. To screen for the thermotolerant strains, transformants were cultured in LB liquid medium with 0.5 mM IPTG for 3 h at 378C to induce ATF expression. Cells were then incubated for 2 h at 558C. After heat treatment, cells were plated on LB agar plates and incubated overnight at 378C. The surviving cells were screened again for the thermotolerant phenotype and cell growth was assessed. The growth experiments were performed in 100 ml LB medium containing 0.5 mM IPTG at both 37 and 508C. Cell growth was monitored by measuring the optical den- sity (O.D.) at 600 nm. For the screening of the osmotolerant phenotype, trans- formants were cultured in LB liquid medium with 0.5 mM IPTG for 3 h at 378C. Cells were then plated on minimal A agar plates with 0.6 M NaCl and 0.5 mM IPTG. After 2 days of incubation at 378C, the growing colonies were selected and individually analyzed for their osmotoler- ance. For the screening of the cold-tolerance phenotype, transformants were cultured in LB liquid medium with PAGE 4 OF 10 0.5 mM IPTG for 3 h at 378C. Cells were then plated on LB agar plates with 0.5 mM IPTG. After 2 days of incu- bation at 158C, the growing colonies were selected and individually analyzed for their cold-tolerance. Plasmids that encoded ATFs were rescued from the selected cells that showed desirable phenotypes, and the ATF sequences of the rescued plasmid were conﬁrmed by DNA sequencing. The plasmids that contained the ATFs were retransformed back into E. coli to conﬁrm the phe- notypic changes. DNA microarray analysis of the thermotolerant cells E. coli cells transformed with either a plasmid encoding the T2 ATF or the pETtac vector were cultured in LB liquid medium with 0.5 mM IPTG at 508C. Total RNA was isolated from exponential-phase cultures with an RNeasy mini kit (QIAGEN, Valencia, CA) and RNAprotect Bacteria Reagent (QIAGEN), and the RNA preparations were subjected to reverse transcription. cDNA labelled with either Cy3-dUTP (C, wild-type E. coli transformed with a control plasmid) or Cy5-dUTP (LT2 and HT2, cells expressing the T2 ATF on low- and high- copy plasmids, respectively) was synthesized from each preparation of total RNA by random priming. Labelled cDNA probes were puriﬁed and hybridized to a DNA microarray slide (TwinChipTM E. coli chip, Digital Genomic, Seoul, Korea) that contained the complete E. coli genome. The slides were scanned by Scanarray lite (Packard Bioscience, Boston, MA) and analyzed by the GenePix Pro 3.0 software (Axon Instrument, Union City, CA). RESULTS Engineering of CRP as an effector domain A potent eﬀector domain is needed to construct ATFs capable of carrying out transcriptional activation or repression of a gene(s). Therefore, we examined the tran- scriptional activities of wild-type CRP and several CRP derivatives in order to identify eﬀective eﬀector domains for the generation of ATFs (Supplementary Table 1). Wild-type CRP (amino acid residues 1–209), CRP D1 (residues 1–180), CRP D2 (residues 137–190), CRP D3 (residues 137–180) and CRP D4 (residues 151–168) were selected as candidate eﬀector domains for our novel ATFs (Figure 1A). These selected CRP eﬀector domains were then each fused to a DNA-binding domain that consisted of 3 ZFs (Z23, Z18 and Z18, ordered in the N- to C- terminal direction) and binds speciﬁcally to the 9-base pair (bp) DNA sequence 50-GCG GCG GGG-30 (Supple- mentary Table 2). We used a reporter plasmid in which the EGFP gene was used as a reporter to select the most potent eﬀector domains. Two diﬀerent reporter plasmids were con- structed by inserting the target DNA sequences for the 3-ZF domain into two diﬀerent regions of the reporter gene. Speciﬁcally, the resulting plasmids were pEGFP-A, which contained the 3-ZF target sequence upstream of the reporter promoter ( 67 to 50), and pEGFP-R, which