The oligodeoxynucleotides used as PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, USA), and Hokkaido System Science (Sapporo, Japan) in purified forms. VCSM13 was obtained from Agilent Technologies (Santa Clara, CA, USA). The 1 kbp ladder DNA size marker was from GeneDireX (Taipei, Taiwan).
Construction of M13-based DNA for arabinose-inducible phage production
The sequence from the ribosome binding site to the HincII site of the M13 phage pII gene was amplified by PCR, using VCSM13 as the template and the primers pII RBS Fw (5′-TTTGGATCAACCGGGGTACATATGA) and pII(HincII) Rv (5′-ATAGTAGTAGCGTTAACATCC). The araBAD promoter sequence with the araC gene was amplified by PCR, using E. coli BL21(DE3) genomic DNA as the template and the primers pBAD Fw (5′-CCCCCCCCCCCTGCATGCATAATGTGCCTGTCAAA) and Rv (5′-CCCGGTTGATCCAAAAAAACGGGTATGGAG). These two PCR fragments were assembled with PstI- and HincII-digested VCSM13 by using the GeneArt Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific, Waltham, MA, USA), and the resultant plasmid was named VCSM13(PBAD-pII).
Two DNA fragments around the M13 intergenic sequence were amplified by PCR, using VCSM13 as the template and the primer sets M13dPS Fw (5′-AAGCGAATT CGCAGCGTGACCGCTACACT) plus M13IGNaeI Rv (5′-TTGACGGGGAAAGCCGGCGA) and pIV-PsiI Fw (5′-TGGCCTCACTGATTATAAAA) plus M13dPS Rv (5′-GCTGCGAATTCGCTTAATGCGCCGCTACAG), and then the two fragments were combined by overlap extension PCR. The DNA fragment digested by PsiI and NaeI was ligated with VCSM13(PBAD-pII) digested by the same enzymes to remove the packaging signal in the M13 intergenic sequence, and the resultant plasmid DNA was named VCSM13ΔPS(PBAD-pII). E. coli HB101 was transformed by VCSM13ΔPS(PBAD-pII).
Phage production by arabinose-induction
E. coli HB101 bearing VCSM13ΔPS(PBAD-pII) was transformed with 1 ng of pBS189R-BsmBI or pSB189L-BsmBI (containing the M13 intergenic sequence) , and plated onto LB agar plates containing 100 μg/mL ampicillin, 25 μg/mL kanamycin, and 0.2% glucose. A single colony was inoculated into 2 mL of LB containing 100 μg/mL ampicillin, 25 μg/mL kanamycin, and 0.2% glucose, and cultured overnight. The overnight culture was diluted 50-fold in 2 × YT medium containing 100 μg/mL ampicillin and 25 μg/mL kanamycin, and cultured at 37 °C with shaking until the optical density at 610 nm (OD610) reached the targeted values. Arabinose was added to the culture at final concentrations of 0.002, 0.02%, or 0.2%, and the culture was incubated further at 37 °C overnight with shaking. For middle-scale phagemid isolation, a 50 mL culture in a 200 mL baffled flask was used. For small-scale phagemid isolation, a 7 mL culture in an 18 × 180 mm test tube was used.
Phage production by VCSM13 infection (conventional method)
A single colony of E. coli JM109/F′ bearing pBS189KR-BsmBI or pSB189KL-BsmBI was inoculated into 2 mL of LB containing 100 μg/mL ampicillin and cultured overnight. The overnight culture was diluted 1000-fold in 2 × YT medium containing 100 μg/mL ampicillin, and cultured at 37 °C with shaking until it reached the targeted OD610 value. VCSM13 helper phage was added at a multiplicity of infection (MOI) of 20:1, and the culture was continued at 37 °C for 2 h with shaking. After phage infection, kanamycin was added at a 25 μg/mL final concentration, and the culture was incubated further at 37 °C overnight with shaking.
Determination of single-stranded DNA yield
The phage particles precipitated by 20% polyethylene glycol (PEG)-6000/2.5 M NaCl from 1 mL of each culture were resuspended in 50 μL of TE (pH 8.0) containing 1 μL of ≥600 mAnson units/mL proteinase K (TaKaRa, Kusatsu, Japan) and 0.1% SDS, and then incubated at 42 °C for 1 h. One microliter of each sample was electrophoresed on a 1% agarose TAE gel, and the DNA was stained by GelRed Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA, USA).
Middle-scale single-stranded DNA purification on an anion-exchange column
The phage particles produced from a 50 mL culture were precipitated by adding a one-tenth volume of 20% PEG-6000/2.5 M NaCl solution to the supernatant of the overnight culture. The solution containing the phage precipitate was centrifuged at 12,000 rpm for 15 min at 4 °C to concentrate the phage. The pellet was resuspended in 1 mL of 10 mM Tris-HCl (pH 8.0), and then 10 μL of 300 mM MgCl2, 5 units of recombinant DNase I (TaKaRa) and 10 mg of RNase A (Nacalai Tesque, Kyoto, Japan) were added, followed by an incubation at room temperature for 1 h to remove contaminations of bacterial nucleic acids in the supernatant. Afterwards, 6.6 μL of 0.5 M EDTA (pH 8.0) was added to the suspension, and then 5 μL of ≥600 mAnson units/mL proteinase K and 50 μL of 10% SDS were added, and the mixture was incubated at 50 °C for 1 h. After this incubation, 0.5 mL of Buffer P3 (QIAGEN, Venlo, Netherlands) was added, and the solution was then centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was applied to a QIAGEN-tip20 column pre-equilibrated with Buffer QBT, and the column was washed with 2 mL of Buffer QC twice. The ss DNA was eluted with 1.6 mL of Buffer QF (QIAGEN) pre-warmed to 50 °C, and then precipitated and resuspended in an appropriate volume of solution.