# Role of Hibernation Promoting Factor
## 4 Materials and Methods  
### Strains  
_P. aeruginosa_ PAO1 and its derivatives were used in these studies. Mutant strains included _P. aeruginosa_ PAO1 _\(\Delta\)lnpf_[21], _P. aeruginosa_ PAO1 _\(\Delta\)lnpf_ + _lpf_, which carries the wild type copy of _hpf_ on the IPTG-inducible vector pMF54 [21,49], and _P. aeruginosa AnelAAspoT_[50]. For overexpression of the 6X-poly-histidine fusion proteins from plasmid pET28a, _E. coli_ BL21 (DE3) was used. PAO1::P_hpf_-HPF-YFP, PAO1::P_BAD_-L5-YFP, PAO1::P_BAD_-L5-6 xHis, and PAO1::P_BAD_-S13-YFP were used for antibody verification experiments. PAO1::P_hpf_ -HPF-YFP was also used in growth and _hpf_ gene expression experiments [41]. **Media and buffers. ** Tryptic Soy Broth and Tryptic Soy Agar (Difco, Sparks, MD, USA) were used for general cultivation of _P. aeruginosa_ and _E. coli_. Isopropyl \(\beta\)-d-1-thiogalactopyranoside (IPTG) (1 mM), carbenicillin (150 \(\upmu\)g/mL) and kanamycin (30 \(\upmu\)g/mL) were added to media when necessary. MOPS buffer contained: 40 mM 3-N-morpholino-propanesulfonic acid (MOPS), 4.0 mM tricine, 0.01 mM FeSO4, 0.276 mM K2 SO4, 0.5 \(\upmu\)M CaCl2, 0.02 mM MgCl2, 50 mM NaCl, 132 mM K2 PO4, pH 7.2. MOPS minimal medium contained MOPS buffer plus 20 mM NH4 Cl and 100 \(\upmu\)L/L micronutrients, supplemented with either 40 mM glucose or 40 mM fructose. The micronutrient solution contained 0.146 mM ammonium molybdate, 20.05 mM boric acid, 1.51 mM cobalt chloride, 0.481 mM cupric sulfate, 0.404 mM manganese chloride, and 0.487 mM zinc sulfate. Phosphate buffered saline (PBS) contained 0.127 M NaCl, 7.0 mM Na2 HPO4, and 3 mM NaH2 PO4 (pH 7.0). **Plasmid constructs. ** For purification of ribosomal proteins, the genes for _rplE_ and _rpsM_ (encoding ribosomal proteins L5 and L13) were cloned into the poly-histidine expression vector pET28a to produce C-terminal 6x-poly-histidine tags, and _hpf_ was purified using the 6x-poly-histidine tag as described previously [22,51]. Restriction endonucleases and DNA ligase were purchased from New England Biolabs (Ipswich, MA, USA). PCR was used to amplify the three genes from _P. aeruginosa_ genomic DNA using the primers: _hpf_-forward CGC ATA TGC AAG TCA ACA TCA GTG GCC ATC, _hpf_-reverse AGC TCG AGT CAG CGCG CCT ACG CCT TGC5; L5-forward GGC ATA TGG CAC GAT TGA AAG AAA TTT ATC and L5-reverse CGC TCG AGC TCG CGT TTC ATC ATG CTC TC; S13-forward GGC ATA TGG CCC GTA TTG CAG GCG TCA AC and S13-reverse GGC TCG AGC AGC TTT TGC CAT GAC TAG. PCR products were ligated in-frame with the 6x-poly-histidine tags using the _Ndel_ and _Xlo1_ restriction sites. **Protein expression and purification. ** 6x-poly-histidine tagged L5 and S13 were expressed and purified from _E. coli_ BL21 (DE3) cells containing pET28a-based plasmids. LB medium (Difco, Sparks, MD, USA) (1 L) supplemented with 30 \(\upmu\)g/mL kanamycin was inoculated with 10 mL of overnight cultures, then incubated at 37 \({}^{\circ}\)C with shaking at 200 rpm for 8 h. After 8 h, 1 mM IPTG (Isopropyl \(\beta\)-D-1-thiogalactopyranoside) was added, and the cultures were incubated for an additional 16 h. Cell pellets were harvested by centrifugation at 3000\(\times\)\(g\) for 15 min at 4 \({}^{\circ}\)C, then frozen at -20 \({}^{\circ}\)C. Cell pellets were resuspended in 10 mL of lysis buffer (100 mM Tris-HCl pH \(\sim\) 8, 150 mM sodium chloride, 5 mM imidazole) and disrupted by sonication with a Branson Digital Sonifier (Branson Ultrasonics, Brookfield, CT, USA) on ice. Cell lysates were then centrifuged at 10,000\(\times\)\(g\) at 4 \({}^{\circ}\)C for 30 min to remove cellular debris. The supernatants containing the soluble proteins were collected and purified by cobalt affinity chromatography on HisPurTM Cobalt Resin (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions for native conditions. 6x-poly-histidine tagged proteins were eluted with Elution Buffer containing 50 mM sodium phosphate, 300 mM sodium chloride, and 300 mM imidazole at pH 7.4 in ten 2 mL fractions.