T leads to accumulation of the recombinant peptides around the colony

T leads to accumulation of the recombinant peptides around the colony and allows for easy library screening. The screening assay used in this study was a modified version of the standard agar diffusion method as previously described [20]. Library-transformed E. coli colonies are overlaid with a tester strain and incubated overnight to allow for peptide expression and tester strain growth. Next day, the plates are inspected for the formation of clear growth inhibition zones around E. coli colonies, which is an indication of active AMP production by the host colony.Step 5: Sequencing of the positive clones to identify AMPs. To identify AMP sequences responsible for activity, E.coli colonies that are in the center of clear zones are selected and either cultured individually (for Sanger-sequencing) or grouped together based on the size of the inhibition zone (for highthroughput sequencing). Their plasmids are extracted and the peptide sequences are identified by DNA sequencing and in silico translation.Application to Discovery of Novel Plantaricin-423 DerivativesPeptide and oligonucleotide library design. Based on the fact that the C-terminal region of Class IIa bacteriocins is much more diverse compared to their N-terminal region and believed to be responsible for antimicrobial activity [32], only the C-terminal region of Pln-423 was mutated in this study. A single mutation was introduced at each position, starting at the 18th amino acid, by replacing the wild-type residue with a random amino acid selected from each of six amino acid Pentagastrin groups (positive/hydrophilic, negative/hydrophilic, polar/hydrophilic, hydrophobic, small/ali-Figure 1. Diagram of the five-step process for the construction and screening of AMP libraries. doi:10.1371/journal.pone.0059305.gA New Antimicrobial Peptide Discovery Pipelinephatic, and structural) shown in Table S1. A second mutation was introduced at each remaining position again with one amino acid selected from the same groups. Single and double random deletions were also introduced at the same region of the wildtype peptide. Thus, one set of single and double mutations and one set of single and double deletions resulted in total of 12,208 unique sequences in the library (Data File S1). Each oligonucleotide sequence contained two 20mer primer-binding regions with two restriction enzyme sites, HindIII and EcoRI, 25331948 and a stop codon.Screening of E. coli library for novel Pln-423 variants. For the construction of Pln-423 mutant library in E.coli, the expression system that consists of a periplasmic-leaky E. coli strain JE5505 and the expression plasmid pFLAG-CTS was employed for direct screening of peptide activities. It should be noted here that this plasmid contains ompA secretion signal sequence and cleavage of this sequence results in a serine residue at the N-terminal of all mature peptides. A total of 1.06105 colonies, approximately 8-fold coverage of the library, were screened against Listeria innocua 33090 in five separate screening experiments. L. innocua was previously deemed as a suitable indicator for ITI-007 web pathogenic L. monocytogenes displaying similar bacteriocin sensitivity [33], therefore it was used as a surrogate strain throughout this study. The selection process involved two criteria; 1) the size of the each inhibition zone was compared to that of wild-type Pln-423 as a correlation to anti-listerial activity level, 2) when several colonies formed inhibition zones that are very similar in size and charact.T leads to accumulation of the recombinant peptides around the colony and allows for easy library screening. The screening assay used in this study was a modified version of the standard agar diffusion method as previously described [20]. Library-transformed E. coli colonies are overlaid with a tester strain and incubated overnight to allow for peptide expression and tester strain growth. Next day, the plates are inspected for the formation of clear growth inhibition zones around E. coli colonies, which is an indication of active AMP production by the host colony.Step 5: Sequencing of the positive clones to identify AMPs. To identify AMP sequences responsible for activity, E.coli colonies that are in the center of clear zones are selected and either cultured individually (for Sanger-sequencing) or grouped together based on the size of the inhibition zone (for highthroughput sequencing). Their plasmids are extracted and the peptide sequences are identified by DNA sequencing and in silico translation.Application to Discovery of Novel Plantaricin-423 DerivativesPeptide and oligonucleotide library design. Based on the fact that the C-terminal region of Class IIa bacteriocins is much more diverse compared to their N-terminal region and believed to be responsible for antimicrobial activity [32], only the C-terminal region of Pln-423 was mutated in this study. A single mutation was introduced at each position, starting at the 18th amino acid, by replacing the wild-type residue with a random amino acid selected from each of six amino acid groups (positive/hydrophilic, negative/hydrophilic, polar/hydrophilic, hydrophobic, small/ali-Figure 1. Diagram of the five-step process for the construction and screening of AMP libraries. doi:10.1371/journal.pone.0059305.gA New Antimicrobial Peptide Discovery Pipelinephatic, and structural) shown in Table S1. A second mutation was introduced at each remaining position again with one amino acid selected from the same groups. Single and double random deletions were also introduced at the same region of the wildtype peptide. Thus, one set of single and double mutations and one set of single and double deletions resulted in total of 12,208 unique sequences in the library (Data File S1). Each oligonucleotide sequence contained two 20mer primer-binding regions with two restriction enzyme sites, HindIII and EcoRI, 25331948 and a stop codon.Screening of E. coli library for novel Pln-423 variants. For the construction of Pln-423 mutant library in E.coli, the expression system that consists of a periplasmic-leaky E. coli strain JE5505 and the expression plasmid pFLAG-CTS was employed for direct screening of peptide activities. It should be noted here that this plasmid contains ompA secretion signal sequence and cleavage of this sequence results in a serine residue at the N-terminal of all mature peptides. A total of 1.06105 colonies, approximately 8-fold coverage of the library, were screened against Listeria innocua 33090 in five separate screening experiments. L. innocua was previously deemed as a suitable indicator for pathogenic L. monocytogenes displaying similar bacteriocin sensitivity [33], therefore it was used as a surrogate strain throughout this study. The selection process involved two criteria; 1) the size of the each inhibition zone was compared to that of wild-type Pln-423 as a correlation to anti-listerial activity level, 2) when several colonies formed inhibition zones that are very similar in size and charact.