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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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This paper presents an analysis of phage-displayed libraries of peptides using Illumina. We describe steps for the preparation of short DNA fragments for deep sequencing and MatLab software for the analysis of the results. Screening of peptide libraries displayed on the surface of bacteriophage (phage display) can be used to discover peptides that bind to any target. The key step in this discovery is the analysis of peptide sequences present in the library. This analysis is usually performed by Sanger sequencing, which is labor intensive and limited to examination of a few hundred phage clones. On the other hand, Illumina deep-sequencing technology can characterize over 10(7) reads in a single run. We applied Illumina sequencing to analyze phage libraries. Using PCR, we isolated the variable regions from M13KE phage vectors from a phage display library. The PCR primers contained (i) sequences flanking the variable region, (ii) barcodes, and (iii) variable 5'-terminal region. We used this approach to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using HiSeq single-end Illumina sequencing of these fragments, we acquired over 2×10(7) reads, 57 base pairs (bp) in length. Each read contained information about the barcode (6bp), one complimentary region (12bp) and a variable region (36bp). We applied this sequencing to a model library of 10(6) unique clones and observed that amplification enriches ∼150 clones, which dominate ∼20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. The results suggest that screens based on repeated amplification and small-scale sequencing identify a few binding clones and miss thousands of useful clones. The deep sequencing approach described here could identify under-represented clones in phage screens. It could also be instrumental in developing new screening strategies, which can preserve diversity of phage clones and identify ligands previously lost in phage display screens., (Copyright © 2012 Elsevier Inc. All rights reserved.)