PAGE 3 OF 10 Nucleic Acids Research, 2008, Vol. 36, No. 16 e102 Figure 1. Engineering a CRP-based eﬀector domain for novel ATFs. (A) A schematic representation of CRP that shows the regions of the protein used as eﬀector domains in relation to known structural features. The numbers indicate positions of the amino acid residues (Supplementary Table 1). (B) Activation of the EGFP reporter gene by 3-ZF-CRP fusion proteins in an EGFP-based reporter system. 3-ZF recognizes the target DNA sequence (50-GCG GCG GGG-30) upstream of the tac promoter of the reporter gene ( 67 to 50), and 3-ZF-CRP fusion proteins activate transcription of the EGFP reporter gene upon induction with arabinose. E. coli cells that harbor both the reporter plasmid and the 3-ZF-CRP encoding plasmid were grown in LB medium with 0.2% arabinose (to induce 3-ZF-CRP fusion expression) and without IPTG (to repress EGFP expression) at 378C for 10 h and then analyzed. (C) Repression of the EGFP reporter gene by 3-ZF-CRP fusion proteins in an EGFP-based reporter system. 3-ZF recognizes the target DNA sequence (50-GCG GCG GGG-30) downstream of the tac promoter of the reporter gene (+24 to +41), and 3-ZF-CRP fusion proteins repress transcription of the EGFP reporter gene upon induction with arabinose. E. coli cells that harbored both the reporter plasmid and 3-ZF-CRP encoding plasmid were grown under the same conditions described above, except that 0.5 mM IPTG was added at inoculation (to induce EGFP expression). Expression of the EGFP reporter gene regulated by a 3-ZF-CRP fusion protein is given as relative ﬂuorescence intensity values. All ﬂuorescence intensities were represented as a relative value compared with that of cells that contained the reporter plasmid and a control plasmid that did not encode a 3-ZF-CRP fusion protein and measured in triplicate. ‘+1’ indicates the transcription initiation site. Error bars represent SD.