Systems and methods for amplification and phage display

Abstract

The present invention generally relates to amplification of biological entities, for example, for phage display. In one aspect, members of a library of biological entities are encapsulated in separate compartments (e.g., in separate microfluidic droplets) and amplified. As a specific example, by putting members of a phage display library into microfluidic droplets such that no droplet contains more than one member of the library, the library can be amplified without any substantial changes in growth rates or population distributions, or other artifacts created due to differences in growth rates or amplification between different members of the library. In some cases, the volume of the compartments can be used to control the copy number of a biological entity during amplification. In certain cases, biological entities with different amplification rates can be amplified independently of each other. In some embodiments, the ratio of a rapidly amplifying biological entity to a slowly amplifying biological entity can be controlled. This can be advantageous, for example, in preserving diversity within a library by preventing rapidly amplifying biological entities from outcompeting slowly amplifying biological entities. For example, certain methods and systems of the invention can be useful in situations where preferential amplification of library members can present a problem.

What is claimed is: 
     
         1 . A method of amplifying a library of replicable bacteriophage clones while maintaining diversity of the library, the method comprising:
 a. distributing a library of replicable bacteriophage clones comprising a plurality of distinguishable replicable bacteriophage clones into a plurality of monodisperse microfluidic droplets-such that substantially no more than one replicable bacteriophage clone is contained in each of the monodisperse microfluidic droplets;   and wherein each of the monodisperse microfluidic droplets-further comprises at least one bacterial cell capable of supporting replication of the respective replicable bacteriophage clones; and   b. culturing the library of step (a) for a sufficient period of time such that the host bacterial cell in each of the monodisperse microfluidic droplets replicates to reach substantially the same number therein, and wherein the library of the replicable bacteriophage clones is amplified when the replicable bacteriophage clones present in the respective monodisperse microfluidic droplets infect the replicated bacterial cells therein;   
       wherein the replicable bacteriophage clones in the library have different amplification rates, and wherein the ratio of bacteriophage clones of different amplification rates is maintained upon amplification thereby maintaining diversity of the library of replicable bacteriophage clones upon amplification. 
     
     
         2 . The method of  claim 1 , wherein the number of bacterial cells within each of the monodisperse microfluidic droplets is substantially the same. 
     
     
         3 . The method of  claim 2 , wherein substantially each of the monodisperse microfluidic droplets further comprises at least ten bacterial cells capable of supporting replication of the respective replicable viral clone. 
     
     
         4 . The method of  claim 1 , wherein each of the monodisperse microfluidic droplets further comprises growth or replication media containing substantially the same amounts of nutrients. 
     
     
         5 . The method of  claim 1 , wherein each of the replicable bacteriophage clones replicates to reach a maximum copy number allowed in each of the monodisperse microfluidic droplets. 
     
     
         6 . The method of  claim 1 , wherein the plurality of distinguishable replicable bacteriophage clones are distributed into a plurality of monodisperse microfluidic droplets using at least one microfluidic device. 
     
     
         7 . The method of  claim 13 , wherein the monodisperse microfluidic droplets are suspended in a perfluorinated liquid and stabilized by at least one surfactant. 
     
     
         8 . The method of  claim 1 , further comprising the step of releasing the amplified distinguishable replicable bacteriophage clones within monodisperse microfluidic droplets into one common fluid. 
     
     
         9 . The method of  claim 8 , wherein the step of releasing is performed by adding at least one destabilization agent into the amplified library of step (b). 
     
     
         10 . The method of  claim 8 , wherein the step of releasing is performed by diluting the at least one surfactant. 
     
     
         11 . The method of  claim 8 , wherein the step of releasing is performed in the presence of electric fields. 
     
     
         12 . The method of  claim 1 , wherein the monodisperse microfluidic droplets have an average diameter of at least about 40 nm. 
     
     
         13 . The method of  claim 1 , wherein the monodisperse microfluidic droplets have an average diameter of about 20 nm to about 500 nm. 
     
     
         14 . The method of  claim 1 , wherein the monodisperse microfluidic droplets have an average diameter of no larger than 200 nm. 
     
     
         15 . The method of  claim 1 , wherein the library comprises at least about 100 distinguishable clones. 
     
     
         16 . The method of  claim 1 , wherein the library comprises at least about 1,000 distinguishable clones. 
     
     
         17 . The method of  claim 1 , wherein the library comprises at least about 10 6  distinguishable clones. 
     
     
         18 . The method of  claim 1 , wherein the library comprises at least about 10 9  distinguishable clones. 
     
     
         19 . A method of producing an amplified library of viral clones, the method comprising:
 providing an initial library of replicable genetic packages having a first distribution of growth rates including a first mean and a first standard deviation; and   amplifying the initial library of replicable genetic packages to produce an amplified library of replicable genetic packages having a second distribution of growth rates including a second mean and a second standard deviation, wherein the first mean and the second mean differ by no more than about 10% relative to the first mean and the first standard deviation and the second standard deviation differ by no more than about 10% relative to the first standard deviation.