Bacteriophage T4

Notes:

The procedure for phage propagation is largely specific for each phage and bacterial host. Here we use propagation conditions for T4 phage and Escherichia coli B bacterial host. It is recommended to use appropriated growth and propagation conditions for your choice of phage and host.

Once a sufficiently high titer phage lysate is obtained please proceed to step 3.

It is recommended to only propagate and purify one phage at a time to prevent cross-contamination.

1| Phage plaque assay for determination of titer (Adams, 1959 )
2A| Phage isolation and propagation via plate lysate
2B| Phage propagation via liquid lysate
3| Phage cleanup (0.22 μm filtering and chloroform)
4| Phage concentration and wash via ultrafiltration
5| Endotoxin removal (Morrison & Leive, 1975 (link); Szermer-Olearnik & Boratyński, 2015 (link))
Notes:

This method is adapted from Szermer-Olearnik & Boratyński (2015) (link), which demonstrates the efficient removal of endotoxins from bacteriophage lysates using water immiscible solvents that are subsequently removed via dialysis. For detailed explanation of the methodology please see Morrison & Leive (1975) (link) and Szermer-Olearnik & Boratyński (2015) (link).

Our adapted method uses a speed vacuum to remove residual organic solvent from phage lysates, instead of the lengthy dialysis washes with similar efficiency.

This step is optional. If you do not require removal of bacterial endotoxins from your phage preparations please go to step 7.

6A| Dialysis removal of organic solvent (Szermer-Olearnik & Boratyński, 2015 (link))
Notes:

This method is adapted from Szermer-Olearnik & Boratyński (2015) (link) and describes the removal of residual organic solvents from phage lysates by dialysis.

Residual organic solvents disable downstream Pierce™ LAL Chromogenic Endotoxin Quantitation assays and must be removed in order to accurately quantify endotoxin concentrations.

Due to the ionic concentration of phage SM buffer used you may end up with greater than the starting volume.

6B| Speed vacuum removal of organic solvent
Notes:

This method is a faster alternative to the dialysis method for the removal of residual organic solvents from phage concentrates.

7| Phage bank storage

Overnight cultures of bacteria (E. coli MG1655 harboring pSG1-EcRADAR or pSG1-CrRADAR plasmid) or negative control (E. coli MG1655 with the pSG1 plasmid) were diluted 1:100 in 60 mL of MMB medium and incubated at 37°C while shaking at 200 rpm until early log phase (OD₆₀₀ of 0.3). 10 mL samples of each bacterial culture were taken and centrifuged at 4000 rpm for 5 min at 4°C. The pellets were flash frozen using dry ice and ethanol. The remaining cultures were infected by phage T4 or T2, at a final MOI of 2. 10 mL samples were taken throughout infection at 0, 15, 27 and 120 min post infection (for EcRADAR), or 0, 15, 27 and 60 min post infection (for CrRADAR), and centrifuged and flash frozen as described above. RNA extraction was performed as described previously.⁵² (link) Briefly, frozen pellets were re-suspended in 1 mL of RNA protect solution (FastPrep) and lysed by Fastprep homogenizer (MP Biomedicals). RNA was extracted using the FastRNA PRO blue kit (MP Biomedicals, 116025050) according to the manufacturer’s instructions. DNase treatment was performed using the Turbo DNA free kit (Life Technologies, AM2238). RNA was subsequently fragmented using fragmentation buffer (Ambion-Invitrogen, cat. #10136824) at 72°C for 1 min and 45 s. The reactions were cleaned by adding ×2.5 SPRI beads (Agencourt AMPure XP, Beckman Coulter, A63881). The beads were washed twice with 80% ethanol and air dried for 5 min. The RNA was eluted using water. Ribosomal RNA was depleted by using the Ribo-Zero rRNA Removal Kit (Epicentre, MRZB12424). Strand-specific RNA-seq was performed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, E7420) with the following adjustments: all cleanup stages were performed using ×1.8 SPRI beads, and only one cleanup step was performed after the end repair step. Following sequencing on an Illumina NextSeq500, sequenced reads were demultiplexed and adapters were trimmed using ‘fastx clipper’ software with default parameters. Reads were mapped to the bacterial and phage genomes by using NovoAlign (Novocraft) v3.02.02 with default parameters as previously described.⁵² (link) Reads mapped to rRNA genes were discarded. Reads mapping equally well to multiple positions in the reference genome, as well as reads containing insertions and deletions as compared to the reference genome, were also discarded. Only reads mapping to the antisense strand of annotated genes were used for the mutation analyses, as these reads represent cDNA generated from the mRNA. Mutations from reference genomes were identified and quantified by counting each mismatch across the transcriptome. Frequency of mismatches was compared between control and RADAR samples throughout the infection time course.

Primer extension analyses were performed as previously described by Pacheco-Sánchez et al. [52 (link)]. An oligonucleotide complementary to the coding strand of the tagB1 gene (P45, Table S3) was ³²P labelled at its 5′ ends in a 10 µl final volume that contained 1 µl 10× buffer, 10 pmol oligonucleotides, 1 µl [γ-³²P]ATP (6000 mCi mmol⁻¹), and 1 U phage T4 polynucleotide kinase. The reaction mixture was incubated for 1 h at 37 °C and 10 min at 70 °C to inactivate the kinase, and the labelled oligonucleotide was filtered through a Bio-Rad Micro Bio-Spin column to eliminate unbound nucleotide. Labelled primers were annealed to total RNA isolated as described above in a 10 µl annealing mixture that contained 2 µl 5× annealing buffer, 10⁵ c.p.m. of 5′-end-labelled primer and 50 µg total RNA template. The mixture was heated at 95 °C for 3 min, incubated at 65 °C for 5 min, and then slowly cooled to 44 °C. cDNA was synthesized by the addition of 40 µl of reverse transcriptase buffer, 1 mM of dNTPs, 0.4 U µl⁻¹ of RNase inhibitor and 8 U of AMV reverse transcriptase. The mixture was incubated for 1 h at 44 °C and the reaction was terminated by adding 5 µl 3 M sodium acetate and 150 µl ethanol. The product of reverse transcription was analysed in urea–polyacrylamide sequencing gels. The gel was exposed to a GS-525 Molecular Imager (Bio-Rad).

We simplified the ACE-seq protocol using the modified Illumina-compatible adapters described above. First, the modified adapters were ligated to 50 ng sheared DNA using the NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^® (New England Biolabs; Catalog #E7645S) according to the manufacturer’s protocol. The adapter-ligated DNA was then treated with T4 Phage β-glucosyltransferase (New England Biolabs; Catalog #M0357S) and UDP-Glucose in 1X NEBuffer supplied with the enzyme in a total reaction volume of 16 μL at 37 °C for 1 h. Next, 4μL formamide was added, and the mixture was incubated at 80 °C for 10 min and then immediately put on ice. The denatured DNA was then subjected to enzymatic deamination using a NEBNext^® Enzymatic Methyl-seq Kit (New England Biolabs; Catalog #E7120S). The DNA was then amplified with six cycles of PCR using NEBNext Q5U Master Mix supplied in the NEBNext^® Enzymatic Methyl-seq Kit. Libraries were sequenced on the Illumina Novaseq6000 platform to generate paired-end data.