Uniform amplification of phage with different growth characteristics in individual compartments consisting of monodisperse droplets.

Uniform amplification of a mixture of phage clones is central to the selection of peptides and proteins presented on the coat proteins of phage (phage display). Uniform amplification cannot be achieved when phage having different rates of growth compete with each other in a common solution. Here we describe a method for uniform amplification of individual phage clones, from a mixture of clones possessing different growth characteristics. We use a microfluidic droplet generator to separate individual clones from a mixture of slowly growing (S) and rapidly growing (R) M13 filamentous phage into droplets of growth media (ca. 200 mm in diameter) containing E. coli. At sufficiently low concentrations of phage, each droplet contains one or no phage particles. Different phage cannot compete for bacterial hosts when isolated in different droplets, and the relative number of S and R clones present at the start is preserved after amplification. Because amplification of phage clones depends on the size of the droplets in which they reside, the use of droplets of uniform size is essential for the success of this process. Display of random peptide sequences on the coat proteins of bacteriophages makes it possible to generate libraries of peptides of great diversity (> 10); rounds of selection and amplification make it possible to select peptides that bind usefully to many targets. Modifications of phage coat proteins, however, can influence the rates of infection of bacteria, the rates of assembly of the new phage particles, or the rates of their production from infected bacteria. When phage with different compositions of the coat proteins (and thus different rates of amplification) compete for the same pool of bacteria, clones that replicate more rapidly capture an increasing fraction of the total pool of bacteria. Amplification of libraries of phage leads to selection of the clones that amplify faster than the rest of the library; it decreases the diversity of the library and leads to loss of potentially valuable clones. For example, a library of 10 diverse clones can contain 10–10 of clones displaying peptides that have similar affinities for a target; but rounds of selection and amplification are likely to retain only a few that bind to target and amplify rapidly. The problem of amplification-based selection is exacerbated when the target has many potential binding sites (e.g. cells, organs). Elimination of undesired competition between different phage clones during amplification would enable selection of wider repertoire of targetbinding phage independently of their relative rates of replication. We demonstrate a procedure that accomplishes this elimination. Our demonstration used non-lytic M13 filamentous phage; this phage uses E. coli as a host, and produces a burst of ca. 1000 plaque-forming units (pfu) of phage within an hour of bacterial infection. To model competition between phage clones, we used two sets of clones: 1) a commercially available library of M13 phage that was engineered to present a 12-mer peptide on the PIII coat protein, and 2) a wild-type (wt) phage (also known as “environmental phage”). Because wt infects bacteria more effectively than engineered phage, wt is an excellent model of a “rapidly growing phage” (R), while the engineered library provides a model of a “slowly growing phage” (S). In our experiments, S phage (exclusively) contained a galactosidase reporter, and formed blue plaques in bacterial colonies on solid media containing the colorimetric galactosidase substrate X-gal. Reporter-free R phage forms clear plaques in the same conditions. During growth of a mixture of S and R phage in a common suspension of E. coli (Figure 1a), the first burst of progeny from the R phage appeared sooner than that for S (Figure 1b). The R/S ratio reaches 170 130 in amplification from 50000 to 10 pfumL , and 580 380 in amplification from 500 to 10 pfumL 1 (Figure 1c). In contrast, when R and S phage were amplified in separate solutions, they both reached a limiting concentration of (3–5) 10 pfumL , maintaining the original ratio of 1:1. Polydisperse emulsions are widely used as compartments for biochemical application. However, in amplification that proceeds to saturation (Figure 1b), different total numbers of phage are produced in compartments of different volumes. To ensure that each phage clone is amplified by the same amount, we placed them inside monodisperse droplets of growth media, generated by a microfluidic flow-focusing device (MFFD), suspended in a perfluorinated liquid, and stabilized by a biocompatible fluoro surfactant (Figure 2a,b). We mixed phage and bacteria, and generated [*] Dr. R. Derda, S. K. Y. Tang, Prof. G. M. Whitesides Department of Chemistry and Chemical Biology, Wyss Institute for Biologically Inspired Engineering, Harvard University 12 Oxford Street, Cambridge, MA 02138 (USA) Fax: (+1)617-495-9857 E-mail: [email protected] [email protected]