Uniform phage display amplification in droplets
Method for uniform amplification of mixtures of phages with different replication rates
The display of random foreign peptide sequences on the coat proteins of bacteriophages ("phage display") is a widely-used research and drug discovery technique for creating and screening highly diverse peptide libraries for peptide sequences that bind usefully to targets. The technique involves rounds of selection (exposure of the phage library to a target) and amplification (pooled replication of target-binding phage clones in host bacteria). However, since different phage clones may have different replication rates when competing for the same pool of bacteria, the amplification stage tends to favor the selection of the fastest replicating clones at the expense of slower-replicating, but potentially valuable, ones. Elimination of undesired competition between different phage clones during amplification would enable selection of a wider repertoire of target-binding phage. Researchers in the laboratory of George Whitesides have developed a non-competitive phage display amplification method that eliminates the influence of the relative rates of replication of different phage clones. This method should expand the repertoire of ligands that can be identified using a wide variety of in vitro selection procedures.
Innovations and Advantages
A microfluidic flow-focusing droplet generator is used to separate individual phage clones from a mixture of both slowly growing (S) and rapidly growing (R) phage into monodisperse droplets of growth media (ca. 200 mm in diameter) containing host bacteria, suspended in a surfactant carrier fluid that prevents the individual droplets from coalescing when collected. At sufficiently low concentrations of phage, each droplet contains one or no phage particles and about 100 bacteria. The droplets in surfactant are collected in a standard Petri dish and rocked at 36 degrees Celsius. By isolating different phage in different droplets, 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.
Uniform Aplification of Phage: a) Isolation of phage in separate droplets that contained bacteria eliminates competition during amplification. b) Images of droplets generated in a flow-focusing microfluidic droplet generator. c) Images of bacteria (arrow heads) and dividing bacteria (arrow) in a droplet. d) R/S ratio obtained by amplification of a 1:1 mixture of R and S phage in bulk solution or in droplets. The number of phage obtained by amplification of phage at different initial concentrations is compared. e) The final concentrations of R and S were proportional to the initial numbers of R and S phage.
This strategy of preserving the ratio between mixtures of different clones by isolating them from each other in individual compartments of identical size is applicable to any in vitro selection/amplification process where preferential amplification of library members is a problem (e.g., phage-, ribosome-, RNA-, and DNA-display, aptamers). Laboratories currently without microfluidic capabilities should find it relatively straightforward to incorporate this fairly simple method into the amplification stage of their procedures.
Intellectual Property Status: A patent application is pending.
R Derda, SKY Tang, and GM Whitesides, “Uniform Amplification of Phage with Different Growth Characteristics in Individual Compartments Consisting of Monodisperse Droplets”, Angew. Chem. Int. Ed. 2010, 49, 5301-5304.
Wiley InterScience Journals #123563036.
Tang, Sindy K. Y.
Whitesides, George M.
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Reference Harvard Case #3733