e102 Nucleic Acids Research, 2008, Vol. 36, No. 16 activation domains. Because of the existence of these var- ious domains (20), CRP could be engineered and used as a transcriptional eﬀector domain to construct novel ATFs. In this study, we selected 40 human ZFs with diverse DNA-binding speciﬁcities and combinatorially assembled them to construct a library of DNA-binding domains that consisted of 3 ZFs each. These DNA-binding domains were then fused to a CRP eﬀector domain to create a fusion protein that either activated or repressed transcrip- tion regardless of the presence of endogenous transcrip- tion factors. Introduction of these novel ATFs into E. coli induced various phenotypic changes such as resistance to heat shock, osmotic pressure and cold shock. The bacte- rial strains were selected that displayed the newly acquired industrially important traits and the genes associated with the selected phenotypes were identiﬁed and characterized. MATERIALS AND METHODS Strains and plasmids E. coli XL1-Blue (Stratagene, La Jolla, CA) and E. coli K-12 MG1655 were used for this experiment. To test an EGFP-based reporter system, MG1655DaraBAD was con- structed by -Red-mediated markerless deletion (22). For complementation assays, the genes related to the thermo- tolerance phenotype, cpxP, ompW and the marRAB operon were deleted from the MG1655 E. coli genome by the above method, producing MG1655DcpxP, MG1655DompW and MG1655DmarRAB, respectively. The T7lac promoter of the pETDuet-1 vector (Novagen, San Diego, CA) was replaced by either a tac promoter or an arabinose-inducible promoter, generating pETtac and pETara, respectively. The pBR322 origin of the high-copy plasmid pETtac was replaced by P15A origin, generating low-copy plasmid pACYCtac. pETtac was used for the expression of the ATF libraries. pACYCtac was used for the expression of the T2 ATF to assess eﬀects of T2 ATF copy number on the thermotolerance phenotype. In addi- tion, for the complementary assays, the genes related to the thermotolerance phenotype, cpxP and ompW, were ampliﬁed with the polymerase chain reaction (PCR) from the MG1655 genomic DNA, and the ampliﬁed cpxP and ompW genes were cloned into pETtac individu- ally or together, producing pETtac-cpxP, pETtac-ompW and pETtac-cpxP/ompW, respectively. pETara was used for the expression of the 3-ZF-CRP fusion proteins, which consisted of 3 ZFs fused to various CRP derivatives, to ﬁnd the most eﬀective CRP eﬀector domain for this study (Figure 1A and Supplementary Table 2). Selection of a potent CRP effector domain with an EGFP-based reporter system Reporter plasmids (pEGFP-A and pEGFP-R) were con- structed by inserting a tac promoter sequence containing the target DNA-binding site for the 3-ZF-CRP fusion proteins and the gene encoding enhanced green ﬂuorescent protein (EGFP) into pACYC184 (New England Biolabs, Beverly, MA). pEGFP-A was constructed by inserting 2 copies of the target DNA sequence (50-GCG GCG GGG-30) upstream of the tac promoter (between positions PAGE 2 OF 10 67 to 50) of pACYC184, followed by insertion of the EGFP gene downstream of the promoter. pEGFP-R was constructed by inserting 2 copies of the target DNA sequence (50-GCG GCG GGG-30) downstream of the tac promoter (between positions +24 to +41) of pACYC184, followed by insertion of the EGFP gene downstream of the promoter. To investigate the eﬀector activity of various CRP derivatives, MG1655DaraBAD was co-transformed with either pEGFP-A or pEGFP-R and a plasmid encod- ing a 3-ZF-CRP fusion protein. Activity of the 3-ZF-CRP fusion proteins was analyzed by assessing their ability to activate or repress transcription of the EGFP gene in pEGFP-A and pEGFP-R, respectively. To examine the eﬀector activity of CRP derivatives to activate transcription of the EGFP gene, transformants that harbored both a plasmid that encoded a 3-ZF-CRP fusion protein and pEGFP-A were grown in LB medium with 0.2% arabinose (to induce 3-ZF-CRP fusion protein expression) and without IPTG (to repress EGFP expres- sion) at 378C for 10 h. To measure the eﬀector activity of CRP derivatives to repress transcription of the EGFP gene, transformants harboring both a plasmid that encoded a 3-ZF-CRP fusion protein and pEGFP-R were grown under the same conditions described above, but 0.5 mM IPTG was added at the time of inoculation (to induce EGFP expression). After harvesting cells by cen- trifugation, cell pellets were washed once with TE buﬀer, and the cell densities of the various preparations were made to be equivalent by the addition of varying amounts of the TE buﬀer. Fluorescence of these cell preparations were measured in an SLM-AMINCO 8100 spectroﬂuo- rometer (Spectronic Instruments, Rochester, NY) at an excitation wavelength of 488 nm and an emission wave- length of 507 nm. All ﬂuorescence intensities were repre- sented as relative values compared with that of a preparation of bacterial cells that contained the reporter plasmid and a control plasmid that did not encode a 3-ZF- CRP fusion protein. Construction of an ATF library An ATF library was constructed with 40 ZFs and CRP D1 (Supplementary Table 2). To construct a DNA frag- ment that encodes a ZF, two 66-nt oligonucleotides (oligos), where 19-nt sequences at each 30 end are comple- mentary to each other, are synthesized. These oligos are hybridized and ﬁlled in by T4 DNA polymerase, produ- cing a 113-bp duplex DNA segment. The resulting 113-bp DNA segment has a sequence encoding a ZF in the middle, preceded by an 18-bp linker sequence containing an XmaI site (bold) at the 50 end (50-CCCGGG GAAAAACCGTAC-30), which is frequently found at the junction between ZF-encoding DNA segments (at their 50 ends) in naturally occurring ZF proteins. An AgeI site, an in-frame stop codon (TAA), and an EcoRI site (50-ACCGGTTAAGAATTC-30) were also incorpo- rated into the 30 region of the 113-bp ZF-encoding DNA segment. The 113-bp products of this ﬁll-in reaction were then digested with XmaI and EcoRI, and subsequently cloned into pUC19 (New England Biolabs) digested with the same enzymes.