title: Uniform amplification of phage display libraries in monodisperse emulsions.
abstract: In this paper, we describe a complete experimental setup for the uniform amplification of libraries of phage. Uniform amplification, which multiplies every phage clone by the same amount irrespective of the growth rate of the clone is essential for phage-display screening. Amplification of phage libraries in a common solution is often non-uniform: it favors fast-growing clones and eliminates those that grow slower. This competition leads to elimination of many useful binding clones, and it is a major barrier to identification of ligands for targets with multiple binding sites such as cells, tissues, or mixtures of proteins. Uniform amplification is achieved by encapsulating individual phage clones into isolated compartments (droplets) of identical volume. Each droplet contains culture medium and an excess of host (Escherichia coli). Here, we describe microfluidics devices that generate mono-disperse droplet-based compartments, and optimal conditions for amplification of libraries of different size. We also describe the detailed synthesis of a perfluoro surfactant, which gives droplets exceptional stability. Droplets stabilized by this compound do not coalesce after many hours in shaking culture. We identified a commercially available compound (Krytox), which destabilizes these droplets to recover the amplified libraries. Overall, uniform amplification is a sequence of three simple steps: (1) encapsulation of mixture of phage and bacteria in droplets using microfluidics; (2) incubation of droplets in a shaking culture; (3) destabilization of droplets to harvest the amplified phage. We anticipate that this procedure can be easily adapted in any academic or industrial laboratory that uses phage display.
authors: Department of Chemistry and Alberta Glycomics Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
authors: Wadim L
authors: WL
authors: Matochko
authors: Simon
authors: S
authors: Ng
authors: Mohammad R
authors: MR
authors: Jafari
authors: Joseph
authors: J
authors: Romaniuk
authors: Sindy K Y
authors: SK
authors: Tang
authors: Ratmir
authors: R
authors: Derda
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D061505:Cell Surface Display Techniques / Q000379:methods*; D046508:Culture Techniques; D004655:Emulsions; D004926:Escherichia coli / Q000235:genetics; D005466:Fluorocarbons / Q000138:chemical synthesis; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D019151:Peptide Library; D011092:Polyethylene Glycols / Q000138:chemical synthesis; D013501:Surface-Active Agents / Q000138:chemical synthesis
chemical_list: D004655:Emulsions; D005466:Fluorocarbons; D019151:Peptide Library; D013501:Surface-Active Agents; D011092:Polyethylene Glycols