acidophilus to the reference genome showing that the α-La scFv reported here could be used immediately for future comparative genome studies

on human-derived L. acidophilus for both research and clinical purposes. Conclusions In this paper we demonstrate the power of combining phage antibody selection directly on bacteria with fluorescence this website activated cell sorting and deep sequencing to either enrich, or deplete, bacteria recognized by specific selected antibodies. Using this approach it becomes possible to assemble genomes directly from complex microbiomes without preculture, or to subtract recognized bacterial species from a microbiome to facilitate genomic analysis of the remaining species. This approach has potential to be applied to different species in different and complex microbial communities. Methods Bacterial cultures and media E.coli DH5αF’ was used to propagate phage and E.coli BL21 Gold was used to express recombinant scFvs. E. coli was grown in 2xyT media containing 1% glucose at 37°C. During phage propagation,

ampicillin and LXH254 kanamycin were used final concentrations of 100 and 25 μg/μl, respectively. Lactobacillus spp. (Table 1) were grown in Lactobacilli MRS Broth (BD 288130) with 5% CO2 atmosphere at 37°C with shaking at 250 rpm. Bifidumbacterium spp. (Table 1) and Peptoniphilus asaccharolyticus were grown in Reinforced Clostridial Medium (BD 218081) with anaerobic condition (85% N2, 5% H2 and 10% CO2) at 37°C with shaking

at 250 rpm. After growing Alisertib in vivo for 18–24 hours, cells were washed twice by spinning down at 3000xg for 5 min, resuspension in 10 ml of washing buffer (WB = PBS, BSA 1%, 2 mM EDTA). After the final washing step cells were resuspended in PBS. Panning A 10 ml overnight (ON) culture of L. acidophilus was grown and washed as described above. Cells were diluted in PBS to an OD600 of ~1.0 (approx. 109 cells/ml) and used for Orotic acid immune-tube (Nunc) coating. The coating process consisted of 1 h incubation at 37°C followed by ON incubation at 4°C. The tube was then blocked with 2% skim milk PBS solution (MPBS) for two hours at room temperature (RT). Phage were generated as described previously and 1012 phage particles of our phage display library [36] were blocked for 1 h at RT with MPBS. Phages were then added to the bacteria coated immune-tube and rotated for 30 min at RT followed by 1.5 h standing at RT. Unbound phages were removed by washing the tube with increasing stringency (number of washes were 20, 25, 30 for the 1st, 2nd and 3rd round of selection respectively) with PBS containing 0.05% Tween (PBST) followed by the same number of washing steps with PBS. After the final wash phages were eluted adding 750 μl of 0.1 M HCl solution for 5 min at RT. The solution was then neutralized with 250 μl of 1.5 M Tris-base pH 8.8 solution. This was followed by phage propagation and titration as described in Sblattero et al.