Therefore, the role of HflKC in the λ lysis-lysogeny switch merit

Therefore, the role of HflKC in the λ lysis-lysogeny switch merits further investigations. Methods Plasmids, bacterial strains and phages Plasmid pQKC was constructed by PCR cloning of the hflK and hflC ORFs (not fused, because the genomic region between these two contains the stop codon for hflK and the RBS for hflC) between the BamHI and SalI sites of pQE30 (purchased from Qiagen, contains the phage T5 promoter under the control of a Lac operator). Construction learn more of pKP219 (which contains the cII gene under the lac promoter LacP and a P15A replication origin) has been described earlier [28]. Plasmid pC2C3 (containing the cII and

cIII genes) was constructed in three steps. First, the NdeI-BamHI fragment of pAB905 containing the cIII gene [29] was cloned into pKP07 [28] and was named pLaCIII (containing the cIII gene under LacP). Then the BglII-XhoI fragment of pLaCIII (i.e. the cIII gene along with the LacP) was cloned into the compatible BamHI-XhoI

sites of pKP106 (which already contained the cII gene under LacP) [28]. The resulting plasmid was named pLaC2C3. In the final step the BamHI-BglII selleck inhibitor fragment of pLaC2C3 (containing both cII and cIII under individual LacP promoters) was cloned into the linearized arm of pK109 (having a P15A origin of replication) [30] at the BglII site. For wild type E. coli, the strain MG1655 (F – λ – ilvG rfb-50 rph-1) was used. The strain AK990 [26] (ΔhflKC:: Kan) served as cells with mutant hflKC. The phage strain λcIII 67 was used as the CIII-defective phage. In this strain, a G to T mutation in the 23rd nucleotide of the cIII ORF leads to an alternative structure of Resminostat the cIII mRNA that is incapable of translation [31]. This is one of the most effective cIII mutants [32] and has been used as cIII- by many workers. Purification of proteins For the purification of the HflKC complex, XL1Blue cells carrying pQKC was used and 100 μg/ml of ampicillin was used for selection. 7.5 ml of the overnight saturated culture was inoculated into 750 ml of fresh

M9 medium with the appropriate antibiotic and allowed to grow on a 37°C shaker incubator till the culture O.D. (at 600 nm) was 0.4-0.5. The culture was then cooled to 18°C and induced by 500 μM IPTG, followed by further growth at 18°C with constant shaking (at 100 rpm) for 20 hours. After induction, bacterial cells were recovered by centrifugation at 3000 g for 10 minutes in Sorvall RC5C, using an SA600 rotor, at 4°C. The medium was decanted out and the pellet was washed with 0.9% NaCl and dissolved in 20 ml of lysis buffer (20 mM TRIS-HCl, pH 8.0, 100 mM KCl, 10% glycerol, 5 mM imidazole, 0.5% NP40, bacterial protease inhibitor cocktail (MBI Fermentas) and 200 μg/ml lysozyme). Cells were then lysed by sonication with 5 pulses (at a pulse rate of 10 mV/30 seconds), followed by centrifugation at 26000 g for 30 minutes at 4°C.

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