Supplementary data collection wit...
Supplementary Data Glenna Meister1, Srinivasan Chandrasegaran2, Marc Ostermeier1 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218 2Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 EXPANDED MATERIALS AND METHODS Plasmids Restriction enzymes, T4 DNA ligase, and T4 polynucleotide kinase were purchased from New England Biolabs (Ispwich, MA) and used according to manufacturer���s instructions. Agarose gel electrophoresis and PCR were performed essentially as described (19). Escherichia coli K-12 strain ER2267 [F�� proA+B+ lacIq ��(lacZ)M15 zzf::mini-Tn10 (KanR)/ ��(argF-lacZ)U169 glnV44 e14-(McrA-) rfbD1? recA1 relA1? endA1 spoT1? thi-1 ��(mcrC-mrr)114::IS10] was obtained from New England Biolabs (Ipswich, MA) and was used as a host for DNA cloning experiments and methylation protection assays. Cells were grown in LB media supplemented with chloramphenicol (50 ��g/ml) and/or ampicillin (100 ��g/ml). All DNA primers were obtained from Invitrogen (Carlsbad, CA).
Construction of pDIM-N7 M.EcoHK31IA and pAR M.EcoHK31IB vectors All primers are listed in Table S1 (Supplementary Data). Plasmids were derived from pDIM-N7-MHhaI[1-302] and pAR-MHhaI[29-327] (15). The pAR plasmid had an NcoI site replaced with an NdeI site for cloning purposes. The pET3a-M38 and pET3a-C23 plasmids containing the M.EcoHK31IA and M.EcoHK31IB genes respectively were a gift from P.C Shaw (17). The natural M.EcoHK31IA gene also encodes the M.EcoHK31IB sequence in a different reading frame. The pETa-M38 plasmid contains the M.EcoHK31IA sequence with the M.EcoHK31IB���s start codon silently removed to prevent expression of both fragments from a single vector. This modified M. EcoHK31IA gene was amplified by PCR using HK31IA-for and HK31IA(NcoI)-rev primers. The M. EcoHK31IA fragment was digested with NdeI and NcoI and ligated into a similarly digested pDIM-N7 MHhaI[1- 302] to make pDIM-N7 M.EcoHK31IA. Two additional silent mutations were made to the M.EcoHK31IA fragment to remove AUG codons which could act as an internal translation site for the M.EcoHK31IB fragment. The M.EcoHK31IB gene was amplified with the HK31IB-for and HK31IB���rev primers, digested with NdeI and SpeI, and ligated into a similarly digested pAR plasmid. The truncated M.EcoHK31IB fragments were amplified using the appropriate forward primer and HK31IB-rev to create truncations ranging from 35 to 50 amino acid residues. Forward primers truncated the N-terminus of the M.EcoHK31IB up to the amino acid residue indicated (ex. HK31IB ��35-for removes the first 34 amino acid residues) and appended an ATG start codon immediately preceding the residue (see Figure S1 for
deletion fragments). The amplified truncated fragment was then inserted into pAR plasmid using NdeI and SpeI restriction sites. See Figure S2 for detailed plasmid maps. Construction of pDIM-N7 Tyr123-�� and pAR ��-Tyr456 plasmids for combinatorial library All primer sequences are found in Table S2 in supplementary data. We initially made several modifications to the plasmids and gene sequences to facilitate cloning and the detection of methylation using EagI for restriction endonuclease assays. Both the M.EcoHK31IA and M.EcoHK31IB genes had EcoRI sites silently removed, and the zinc finger sequences for Tyr123 and Tyr456 (20) had NdeI and SpeI sites, which were silently removed. The pDIM-N7 M.EcoHK31IA plasmid also had an EagI restriction site removed (that was located near the intended EagI target site) in order to create a larger digestion fragment that could be detected in restriction endonuclease assays. The target sites were designed with a methylation site (5���-CGGCCG-3���) flanked by the Tyr123 and Tyr456 zinc finger binding sites (Figure 1A). This methylation site is also recognized by the EagI (5���-CGGCCG-3���) and EaeI (5���-YGGCCR-3���) restriction enzymes. The zinc finger DNA binding sites are separated from the methylation site by 0, 1, 2 or 3 base pairs (labeled 0 bp, 1 bp, 2 bp, and 3 bp target sites) with the methylation site centered between the zinc finger binding sites. Oligonucleotides (TyrZF 0 bp Target- for and rev, TyrZF 1 bp Target-for and rev, TyrZF 2 bp Target-for and rev, and TyrZF 3 bp Target-for and rev) containing the zinc finger binding sites and methylation site were annealed, phosphorylated and ligated into the pDIM-N7 M.EcoHK31IA plasmid between the XmaI and EcoRI sites.