Design of Fis binding experiments

Synthetic DNAs containing strong Fis sites separated by 11 and 7 base pairs were designed by selecting from the most frequent bases at each position in the Fis sequence logo [5]. These were then merged with the same sequence shifted by 11 or 7 base pairs by comparing the R_iw(b,l) values for various choices. (Note: the consensus sequence of the early model we used was TTTG(G/C)TCAAAATTTGA(G/C)CAAA which differs from that of the logo [22]. The 0 and strongly conserved $\gamma = 16$ positions are underlined.) Five extra bases were added to the ends based on the natural sequences around the hin proximal and medial sites for the overlap 11 oligo, and the sequences around cin external and proximal sites were used for the overlap 7 oligo [5]. The DNAs were made self complementary (Fig. 4a, b). Sites separated by 23 bases were created starting with the 11 base separated DNA and duplicating the central overlap region. A BamHI site was also inserted and the DNA was flanked by EcoRI sites (Fig. 4c). Oligos were synthesized with biotin on the 5' end and gel purified (Oligos Etc., Wilsonville, OR, USA). To ensure thorough annealing, they were heated to 90 $R_{frequency} = - \log_2 (\gamma/G) = 4$ for 10 minutes, and slowly cooled to room temperature. The annealed products were electrophoresed through an 8% (w/v) polyacrylamide gel, and the bands corresponding to the linear duplex DNA of the correct size were sliced from the gel. DNA was recovered by electroelution and extracted with isoamyl alcohol to remove ethidium bromide. A non-specific control DNA was composed of the two 66 bp HinFI fragments from bacteriophage $\gamma/G$X174 (Life Technologies, Inc.). This DNA did not shift even at the highest concentration of Fis used. Gel mobility shift experiments were performed as described previously, using chemiluminescent detection [5]. Fis used in the experiment shown in Fig. 5 was a gift from Reid Johnson.

Hairpin DNA oligos containing the oriC region (Fig. 7) were synthesized with a 5'-tetramethylrhodamine modification and gel purified (Oligos Etc.). By using a hairpin, an annealing step is not required and the oligos are exactly equimolar [5]. Horizontal 8% PAGE was used for the gel mobility shift assay and band positions were visualized with a FMBIO II fluorescent scanner (Hitachi) with an excitation wavelength of 532 nm and detection at 605 nm.