Phage amplification

The procedure for epitope mapping by the phage display method involves biopanning, amplification, assay for positive colonies, DNA isolation, and sequencing. Some steps of the procedure must be performed at the same time. Therefore, we describe the protocols in Subheadings 3.1. and 3.2. on a day-by-day manner. All steps of biopanning and phage amplification should be carried out under sterile conditions, when possible. 

Bacterial Culture and Phage Amplification and Titering (Methods Outlined in Subheadings 3.3 and 3.4)... 

Cells are seeded onto tissue culture wells 24-48 h before panning. Only a single well is required for each panning round. Once started, each round of the panning procedure requires approximately 4-5 h to complete. Additional time is required for bacterial plating for titer determination and phage amplification. 

On column infection of co//cells with bound phages Phage amplification Phage elution... 

Fig. 13.4 Workflow for a phage-amplification assay using stable-isotope labeled phages to simultaneously determine the presence of Staphylococcus aureus in a sample, as well as the susceptibility of microorganism to two antibiotics—clindamycin and cefoxitin. (Reprinted with permission from Rees et al. (2015), copyright 2015, American Chemical Society)...

A majority of the work done in our laboratory has been conducted on E. coli and B. anthracis using MS2 and gamma-phage, respectively. Amplification results have been obtained from several other host bacteria and bacteriophages. Table 14.1 summarizes the materials studied. In all cases studied, no two bacteriophages contained the same proteins. 

The amplification products are ligated into the phage or phagemid vectors after cutting with the appropriate restriction enzymes... 

A non-contact heating method called infrared-mediated temperature control has been employed for PCR. Because the chip material (polyimide) does not absorb IR, only the solution absorbs IR. Therefore, the low thermal mass of the solution allows for fast thermal cycling, and 15 cycles have been achieved in 240 s Amplification of X phage DNA (500 bp) was first conducted at 94°C for 10 s, followed by 15 cycles of 94°C (2 s), 68°C (2 s), 72°C (2 s), then finally stopped at 72°C for 10 s [192],... 

Amplification and renormalization Amplification and renormalization are combined in the simplified form used in SELEXION. Each phage surviving elution is scaled the same amount, yielding a renormalized library of the same size as the original library, but with the mole fractions that resulted from elution. 

---