Phage protocols

In the phagemid/helper phage system used in this work, virions containing helper phage genomes are referred to as a phage, whereas virions containing phagemid or recombinant phagemid genomes are referred to as Phagemid Particles (PPs).

2.2.3.1 Phage propagation

VCSM13 (gIII+) and VCSM13d3 (∆gIII) helper phage have been used in this work. VCSM13 phage was propagated and titred on strain TG1 (see section 2.2.1), whereas the VCSM13d3 phage stocks were generated on complementing strain K1976 (see section 2.2.1). The VCSM13 phage was used for production of infectious recombinant PPs for panning and affinity binding. The VCSM13d3 phage was used for selection of inserts encoding the secretome protein-c-myc-pIII translational fusions. In the VCSM13d3-mediated display, the secretome protein-c-myc-pIII fusions are displayed in five copies, i.e. the display is ‘polyvalent’, in contrast to a single fusion per virion when the wt helper phage, VCSM13, is used.

All helper phage stocks were derived from a single plaque, using a plate-based (plate)

method, by mixing 105 – 106 phage from a resuspended plaque with 0.2 mL of the appropriate

host strain overnight culture and 2.5 mL of soft agar, and poured over the appropriate solid medium in a Petri dish. The plates were incubated overnight at 37°C and the phage were subsequently extracted from the lawn by overlaying 5 mL of 2×YT media and incubating with slow rotary agitation for 1 h at RT. The extracted phage were collected and separated from the cells by centrifugation at 13,200×g for 20 min at 4 °C, followed by heating at 65 °C for 10 min to kill the remaining viable cells. Phage was additionally purified and concentrated as described in section 2.2.3.2.

Phage stocks of the volumes over 100 mL were obtained using a liquid-based (liquid)

method. An exponentially growing E. coli culture (OD600 nm ~0.2) was infected by a phage stock

(obtained from a single plaque using the plate method) at the multiplicity of infection (m. o. i.) of 50 phage per cell and incubated for 30 min at 37 °C without agitation, then 6 h with shaking at 300 rpm. The cells were pelleted by centrifugation at 13,200×g for 20 min at 4 °C. The supernatant (phage stock) was collected and the remaining bacterial cells were killed by heating at 65°C for 10 min. Phage stocks were additionally purified and concentrated as described in section 2.2.3.3..

2.2.3.2 Preparation of PPs

In those experiments where PPs were produced, the liquid method was mainly used (see section 2.2.3.2.1), except for PPs derived from the large shotgun metagenomic library that were prepared by plate method (see section 2.2.3.2.2).

2.2.3.2.1 PPs preparation by liquid method

Cultures of E. coli cells containing recombinant phagemids or phagemid vectors

(100 – 200 mL) in the exponential phase of growth (OD600 nm ~0.2) were infected at an m. o. i.

of 50 with appropriate helper phage and incubated at 37ºC without shaking for 30 min (for VCSM13) or 1 h (for VCSM13d3).

Infected cells were pelleted by centrifugation at 3,300×g for 10 min at RT to remove the remaining unabsorbed helper phage, resuspended in an equal volume of fresh media and incubated for 6 h at 37ºC with aeration. Host cells were removed by centrifugation at 13,200×g for 20 min at 4 ºC, the supernatant was collected and the PPs were concentrated and purified as described in section 2.2.3.3.

2.2.3.2.2 PPs preparation by plate method

To prepare PPs derived from the large metagenomic library in pDJ01, library cells were, after an overnight amplification, diluted 100-fold into 100 mL of 2×YTCm25 media and grown until exponential phase. Cells in the exponential phase of growth (OD600 nm ~0.2) were

infected with the VCSM13d3 helper phage and harvested by centrifugation as described in section 2.2.3.2.1. The resulting pellet was mixed with 40 mL of soft agar. Agarose-embedded cells were poured over 16 double-layer Cm plates and incubated overnight at 37°C [352]. Both

93 the soft agar and the double-layer plates were prepared as described in section 2.1.2.7 except that molecular biology grade agarose was used instead of bacteriological agar. PPs were extracted from each plate by adding 5 mL of 2×YT media on top of the soft agar surface and incubating with rotary agitation for 1 h at RT. Aliquots of the media containing PPs collected from the individual plates were pooled together and PPs were purified and concentrated (see section 2.2.3.3).

2.2.3.3 Purification and concentration of phage and PPs

The 2×YT medium containing extracted virions was filtered through a sterile Millex®- HV Syringe Filter Units (EMD Millipore, USA) containing a very low protein binding Durapore® (PVDF) membrane with a 0.45 µm pore size to eliminate any remaining bacterial cells. Virions were further concentrated (100 – 200×) by precipitation in pre-chilled phage precipitation buffer (PEG/NaCl; section 2.1.2.6) for 1 h on ice, pelleted at 13,200×g for 30 min at 4 ºC and resuspended in 1 mL 10 mM Tris-HCl (pH 7.6). Virions were titred as described in section 2.2.3.6 and stored at 4ºC short-term or in 7% DMSO at –80ºC long-term.

2.2.3.4 Disassembled virion gel electrophoresis

Disassembled virion gel electrophoresis was used to detect total viral ssDNA (the sum of encapsulated and free viral DNA). Prior to electrophoresis, virions were mixed with SDS-containing buffer (see section 2.1.2.6) in a 3:1 volume ratio (virions to buffer) and disassembled by incubation at 70°C for 20 min. Disassembled virions were directly loaded onto a gel containing 0.6% (w/v) agarose in 1× TAE buffer and subjected to agarose gel electrophoresis as described in section 2.2.2.2. Total viral ssDNA was visualised by post-

staining with a solution of 0.5 µg mL-1 ethidium bromide (EtBr).

2.2.3.5 Native virion gel electrophoresis

Native virion agarose gel electrophoresis allows detection of the free and encapsulated viral DNA, and was used to analyse the stability of recombinant virions and to verify sarkosyl selection. Samples were loaded onto 0.4% agarose gels in BlueJuice™ Gel Loading Buffer (Life Technologies, USA) and electrophoresis was performed at 1 V cm-1 in Wide mini-Sub® Cell GT horizontal electrophoresis system (Bio-Rad) for 17 h. Free (not encapsulated) phage

encapsulated phage ssDNA, virions were subsequently disassembled by soaking the gel in 0.2 M NaOH for 1 h, followed by neutralisation in 0.45 M Tris (pH 7.1) for 15 min and soaking again in EtBr solution to visualise the DNA that became exposed after the virion disassembly step by NaOH.

2.2.3.6 Enumeration of phage and PPs

2.2.3.6.1 Titration of phage and PPs

The phage and PPs were titred using a quick ‘drop’ method for titre estimation, or a full ‘plate’ method for increased accuracy.

In the quick ‘drop’ method, 10 µL drops of serially diluted phage were placed onto a soft agar layer containing 200 µL of TG1 or K1976 overnight culture. Plaques that developed in the area of the absorbed drops were counted to estimate the approximate number of phage. For accurate titres, the appropriate volume of a dilution, such that 200 – 300 plaques per plate are obtained (as estimated by quick ‘drop’ method), was mixed with 200 µL of TG1 or K1976 overnight culture and 2.5 mL of soft agar. The mixture was poured over pre-warmed plates (in triplicate) and incubated overnight at 37°C.

The helper phage were titred as described above, and the titre was expressed as the

number of plaque-forming units per mL (PFU mL-1). VCSM13 phage was titred on TG1 cells

and VCSM13d3 phage on the complementing strain, K1976.

The infectious PPs were titred in triplicate on TG1 as described above, except that instead of counting plaques, the titre was determined through transduction of the Cm resistance

marker (CmR), carried by PPs, into the host strain. For this, specially prepared double-layer Cm

plates (see section 2.1.2.7) were used to allow in-agar infection of the TG1 strain and expression of transduced CmR marker by indicator strain TG1 prior to antibiotic exposure [352]. The PP

titre was expressed as colony-forming units per mL (CFU mL-1).

2.2.3.6.2 Phage and PPs quantification by densitometry

Non-infectious phage and PPs were quantified by densitometry based on the amount of encapsulated DNA [409]. Phage ssDNA, released from the SDS-disassembled virions and subjected to disassembled virion agarose electrophoresis (see section 2.2.3.4), was stained with EtBr and quantified densitometrically. Given that densitometric measurement of band density in agarose gels is based on comparison with a standard curve generated using DNA of known

95 concentrations, every gel contained a series of two-fold dilutions of purified helper phage ssDNA (10 – 360 ng) as standards. The gel was digitally photographed as described in section 2.2.2.2 and densitometric analysis was performed using the Science Lab 2001 Image Gauge software version 4.0 (Fujifilm, Japan) and Excel (Microsoft, USA).

A second-degree polynomial function was used for standard curve fitting over the series of standard data points. Conversion of the calculated amount of ssDNA in the samples into the

number of virions via number of genome equivalents was based on molecular weight of ssDNA

genome, calculated from the size of phage or phagemid DNA and its base composition. The number of genome equivalents corresponds to the number of genomes in a particular PP sample, which may correspond to a single PP, if a single genome is packaged into a particle, or a unit of a PP corresponding to a single genome, if multiple genomes are sequentially packaged into a long PP. The population of PPs obtained using VCSM13d3 helper phage in the presence of functional pIII expressed from the phagemid typically demonstrates a wide distribution of PP lengths, containing from one to several genomes packaged into a single virion, whereas the PPs obtained using the VCSM13 are typically 95% single-genome PPs and 5% of double-genome PPs [354].