Inimize such errors, it is apparent from these findings that the initial template concentration was too high which possibly resulted in multiple template fragments per droplet, causing cross-recombination between fragments, resulting in extra sequences in the final amplicon library. For this particular study, we have employed an E. coli AN-3199 site expression system due to the fact that most Class II-a bacteriocins do not display activities against E. coli. In the case of generating mutants with host toxicity, we presumed that they were simply eliminated from the library during screening as those clones expressing toxic peptides would not grow into colonies. However, based on the activity spectrum of the AMP of interest, a variety of other engineered microbial systems can be utilized as the expression host in this approach, as well as other biological systems to study peptides for their binding affinities (by phage display) or for their cell-penetrating characteristics (by phage or plasmid display). The work presented here enables the production of fully customized libraries containing hundreds of thousands of peptides in a very cost-effective way. As we attempted to demonstrate by small-scale library sequencing, this method can easily be adapted to screening of much larger libraries by employing a highthroughput screening tool 
combined with massively parallel deep sequencing. Robotic colony picking systems such as QPix by Molecular Devices and its “halo recognition” application can be adapted to recognition of growth inhibition zones and picking the center colonies in a high-throughput manner. Integration of such an automated system will eliminate the cumbersome colonypicking process by the researcher and will translate this method to a true high-throughput process capable of routinely producing and screening hundreds of thousands of AMP candidates. This will remarkably accelerate current AMP research towards developing novel therapeutics and biotechnological materials.Methods Construction of the Peptide LibraryThe oligonucleotide library was obtained from Mycroarray (Ann Arbor, MI). The oligonucleotides were amplified by emulsion PCR following the protocol developed by Williams et al. (2006) with some modifications. Briefly, 10 ng of the oligonucleotide library was mixed with a solution containing 100 pmoles of 10457188 each primer, 6 mM MgCl2, 2 mM dNTPs, 0.5 g/l BSA, and 10 units of Phusion Hot Start DNA Polymerase (NEB) in a final volume of 100 ml. The PCR mix was emulsified by addition to 600 ml oil-surfactant mixture and stirring for 10 min at 1000 rpm on a magnetic stirrer in an ice-cooled glass vial. The emulsified reaction mix was dispensed in 50 ml aliquots and the amplification was performed by 30 cycles of 98uC for 15 s, 55uC for 20 s, and 72uC for 20 s. After extraction with two rounds of diethyl-ether and ethyl-acetate and agarose gel-purification, PCR 
products were digested with HindIII and EcoRI (NEB) and ligated into pFLAG-CTS expression vector (JW-74 Sigma-Aldrich) linearized with the same enzymes. Ligation products were transformed into electrocompetent E. coli JE5505 cells (Strain JE5505 was obtained from the Yale University E. coli Genetic Stock Center) and cloning was confirmed by DNA sequencing (University of Michigan Sequencing Core).A New Antimicrobial Peptide Discovery PipelineScreening Assay for AMP ActivityThe screening 26001275 method used in this study was a modified version of the standard colony overlay method as previously described [20.Inimize such errors, it is apparent from these findings that the initial template concentration was too high which possibly resulted in multiple template fragments per droplet, causing cross-recombination between fragments, resulting in extra sequences in the final amplicon library. For this particular study, we have employed an E. coli expression system due to the fact that most Class II-a bacteriocins do not display activities against E. coli. In the case of generating mutants with host toxicity, we presumed that they were simply eliminated from the library during screening as those clones expressing toxic peptides would not grow into colonies. However, based on the activity spectrum of the AMP of interest, a variety of other engineered microbial systems can be utilized as the expression host in this approach, as well as other biological systems to study peptides for their binding affinities (by phage display) or for their cell-penetrating characteristics (by phage or plasmid display). The work presented here enables the production of fully customized libraries containing hundreds of thousands of peptides in a very cost-effective way. As we attempted to demonstrate by small-scale library sequencing, this method can easily be adapted to screening of much larger libraries by employing a highthroughput screening tool combined with massively parallel deep sequencing. Robotic colony picking systems such as QPix by Molecular Devices and its “halo recognition” application can be adapted to recognition of growth inhibition zones and picking the center colonies in a high-throughput manner. Integration of such an automated system will eliminate the cumbersome colonypicking process by the researcher and will translate this method to a true high-throughput process capable of routinely producing and screening hundreds of thousands of AMP candidates. This will remarkably accelerate current AMP research towards developing novel therapeutics and biotechnological materials.Methods Construction of the Peptide LibraryThe oligonucleotide library was obtained from Mycroarray (Ann Arbor, MI). The oligonucleotides were amplified by emulsion PCR following the protocol developed by Williams et al. (2006) with some modifications. Briefly, 10 ng of the oligonucleotide library was mixed with a solution containing 100 pmoles of 10457188 each primer, 6 mM MgCl2, 2 mM dNTPs, 0.5 g/l BSA, and 10 units of Phusion Hot Start DNA Polymerase (NEB) in a final volume of 100 ml. The PCR mix was emulsified by addition to 600 ml oil-surfactant mixture and stirring for 10 min at 1000 rpm on a magnetic stirrer in an ice-cooled glass vial. The emulsified reaction mix was dispensed in 50 ml aliquots and the amplification was performed by 30 cycles of 98uC for 15 s, 55uC for 20 s, and 72uC for 20 s. After extraction with two rounds of diethyl-ether and ethyl-acetate and agarose gel-purification, PCR products were digested with HindIII and EcoRI (NEB) and ligated into pFLAG-CTS expression vector (Sigma-Aldrich) linearized with the same enzymes. Ligation products were transformed into electrocompetent E. coli JE5505 cells (Strain JE5505 was obtained from the Yale University E. coli Genetic Stock Center) and cloning was confirmed by DNA sequencing (University of Michigan Sequencing Core).A New Antimicrobial Peptide Discovery PipelineScreening Assay for AMP ActivityThe screening 26001275 method used in this study was a modified version of the standard colony overlay method as previously described [20.
