H-NS and Fis binding to E. coli K12
H-NS and Fis binding to E. coli K12
The E. coliK-12 MG1655 bacterial strains used in this work are the following: E. coli MG1655 (F- lambda- ilvG- rfb-50 rph-1); MG1655 ?hns (?hns::Kanr); MG1655 ?fis (?fis::Kanr); MG1655 hns-FLAG (hns::3xFLAG::Kanr); MG1655 fis-FLAG (fis::3xFLAG::Kanr). Luria-Bertani (0.5% NaCl) broth and agar (15 g/liter) were used for routine growth. Where needed, ampicillin, kanamycin, and chloramphenicol were used at final concentrations of 100, 30, and 30 ug/ml respectively. ChIP was performed as previously described (Grainger et al, 2004) with some modifications to the protdegree Col. Cells were grown aerobically at 37degree C to the desired OD600 and formaldehyde was added to a final concentration of 1%. After 20 min of incubation, glycine was added to a final concentration of 0.5 M to quench the reaction and incubated for a further 5 min. Cross-linked cells were harvested by centrifugation and washed twice with ice-cold TBS (pH 7.5). Cells were resuspended in 1 ml of lysis buffer (10 mM Tris [pH 8.0], 20% sucrose, 50 mM NaCl, 10 mM EDTA, 20 mg/ml lysozyme and 0.1 mg/ml RNase A) and incubated at 37degree C for 30 min. Following lysis, 3 ml immunoprecipitation (IP) buffer (50 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS] and PMSF [final concentration 1 mM]) was added and the lysate passed through a French pressure cell twice. 2 ml aliquots were removed and the DNA sheared to an average size of ~200 bp using a Bioruptor (Diagenode) with 30 cycles of 30 sec on/off at high setting. Insoluble cellular matter was removed by centrifugation for 10 min at 4degree C, and the supernatant was split into two 800 microl aliquots. The remaining 400 microl was kept to check the size of the DNA fragments.<br><br>Each 800 microl aliquot was incubated with 20 microl Protein A/G UltraLink Resin (Pierce) on a rotary shaker for 45 min at room temperature to get rid of complexes binding to the resin non-specifically. The supernatant was then removed and incubated with either no antibody (mdegree Ck-IP), FLAG mouse mondegree Clonal antibody (Sigma-Aldrich) or RNAP Beta subunit mouse mondegree Clonal (Nedegree Clone) and 30 microl Protein A/G UltraLink Resin, pre-incubated with 1mg/ml bovine serum albumin (BSA) in TBS, on a rotary shaker at 4degree C overnight (FLAG antibody) or at room temperature for 90 min (RNAP Beta subunit antibody). Samples were washed once with IP buffer, twice with IP buffer + 500 mM NaCl, once with wash buffer (10 mM Tris [pH 8.0], 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet P-40 and 0.5% sodium deoxycholate) and once with TE (pH 7.5). Immunoprecipitated complexes were eluted in 100 microl elution buffer (10 mM Tris [pH 7.5], 10 mM EDTA and 1% SDS) at 65degree C for 20 min
Experimental Factor: GENOTYPE
hns::3xFLAG
Experimental Factor: GROWTHCONDITION
stationary
Experimental Factor: IMMUNOPRECIPITATE
FLAG antibody