16. Therefore, it is likely that a cell is infected by only one phage and that the amount of infected bacteria is equal to the amount of the initial phage concentration. After addition of the phages, one aliquot was immediately used for determination of the phage titer. Then, phages were allowed to adsorb NU7026 for 15 min. Afterwards, cultures were diluted in LB 104-, 105-, 106- and 107 -fold and incubated at 37°C for 60 min. Samples for phage enumeration were taken aseptically at different time points after infection. The burst size was determined as: (phage titer at the end of the single step growth curve at time
point 55 min minus phage titer at time point 20 min) divided by phage titer at time point 20 min. The latent phase was estimated at the midpoint of the exponential phase of a one step growth experiment [40, 41]. Sequencing, analysis and annotation of phage genomes To isolate phage DNA, phages were propagated in top-agar plates as described above. After growth at 37°C the plates were overlayed with 10 ml SM buffer and incubated with
shaking at 4°C for 4 h. The supernatant was sterile filtrated (0.22 μm) and stored at 4°C. Phage DNA was isolated using the Qiagen Lambda Kit according to manufacturer’s instructions. Ten ml phage lysate with a titer of at least 1*1010 phages/ml were used to isolate up to 1 μg/μl pure phage DNA. Digestion with restriction endonucleases was done following the protocols selleck products of the manufacturer. Whole click here Genome sequencing of the phage JG024 was done at the McGill University and Génome Québec Innovation Centre (Montréal, QC, Canada) using the Genome Sequencer FLX and 454 Technology. A total of 66,684 reads with an average length
of 344 bases was assembled to one single contig with a 300-fold coverage. The annotation of the unknown phage genes was done by using the software GeneMark.HMM [31]. The Heuristic approach of GeneMark was used to identify genes in small genomes under 100 kb. The identified genes were compared with the NCBI ORF Finder [32]. Nucleotide sequences were scanned for homologues using the Basic Alignment Search Tool (blastx) [26]. To search for tRNA genes Verteporfin ic50 in the phage sequences the internet tool tRNAscan-SE 1.21 was used [29]. Sequence comparison was conducted using ClustalW2 online analysis tool [42]. Investigation of the codon usage was performed using a software tool based on JCat [43]. The genome sequence as well as the annotation is deposited with the GenBank (National Center for Biotechnology Information) using the following accession number: GU815091. Identification of promoter regions, terminator structures and other motifs The genome of phage JG024 was scanned for the presence of sigma 70-dependent promoter regions using the web service SAK [44]. Putative promoter regions with a score above 1 were scanned for the presence of conserved -10 and -35 regions using the Virtual Footprint software [45]. Two promoter regions were identified in this way.
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