Methods Of Identifying Biologically Active Random Peptides In Prokaryotic Cells And Libraries Of Prokaryotic Cells Expressing Candidate Biologically Active Random Peptides

Abstract

The present disclosure provides methods and systems for identifying biologically active random peptides (BARPs) in prokaryotic cells, such as bacterial cells, and libraries of transformed bacterial cells, where each cell/colony expresses a different candidate BARP.

1 . A method for identifying biologically active random peptides (BARPs) in prokaryotic cells, the method comprising:
 providing a library of test nucleic acid sequences, the library comprising a plurality of different test nucleic acid sequences encoding a plurality of candidate BARPs, wherein each test nucleic acid sequence consists of nucleic acids encoding, in the following order: a start codon, a spacer codon selected from alanine or glycine, a first cystine residue, a random sequence of 6-20 amino acids representing a candidate BARP, a second cysteine residue, such that the first and second cysteine residues flank the random sequence of amino acids, and a stop codon, and wherein each test nucleic acid sequence in the library is flanked by recombinatorial cloning primer sequences;   creating a library of recombination vectors from the library of test nucleic acid sequences, wherein each vector comprises a test nucleic acid sequence from the library and a nucleic acid sequence encoding a selectable marker operably linked to the test nucleic acid sequence;   transforming a plurality of phenotypically homogenous prokaryotic cells of the same species with the library of recombination vectors;   screening the prokaryotic cells for the presence of the selectable marker;   selecting prokaryotic cells with the selectable marker to produce a library of recombinant prokaryotic cells, wherein each prokaryotic cell comprises a recombination vector from the library, wherein identification of the selectable marker indicates expression of a candidate BARP by the prokaryotic cell;   screening the library of recombinant prokaryotic cells throughout development for the occurrence of a new phenotype, wherein the new phenotype is discernible from the phenotype of a corresponding wild type prokaryotic cell without the candidate BARP and wherein the presence of the new phenotype indicates the candidate BARP is responsible for the new phenotype; and,   upon observance of a new phenotype, determining the sequence of the candidate BARP from the recombinant prokaryotic cells exhibiting the new phenotype.   
     
     
         2 . The method of  claim 1 , further comprising:
 verifying the new phenotype associated with the BARP by independently transforming additional prokaryotic cells with a vector encoding the BARP; and   screening for the presence of the new phenotype, wherein the presence of the new phenotype in the new transformed prokaryotic cell indicates that the BARP is responsible for the new phenotype.   
     
     
         3 . The method of  claim 1 , wherein the random sequence of amino acids is 6 amino acids in length and wherein each test nucleic acid in the library consists of SEQ ID NO: 1, wherein “n” represents any nucleotide, and wherein each “n” for each test nucleic acid sequence in the library is independently selected. 
     
     
         4 . The method of  claim 1 , wherein the random sequence of amino acids is 12 amino acids in length. 
     
     
         5 . The method of  claim 1 , wherein the prokaryotic cells are bacterial cells. 
     
     
         6 . The method of  claim 5 , further comprising, testing the activity of the candidate BARP in a second bacterial species by independently transforming a plurality of phenotypically homogenous cells of a second species of bacteria with a vector encoding the candidate BARP, and screening for the presence of the new phenotype, wherein the presence of the new phenotype in the transformed bacterial cells of the second bacterial species indicates that the candidate BARP is responsible for the new phenotype and that the candidate BARP is active in a second bacterial species. 
     
     
         7 . The method of  claim 1 , wherein the selectable marker is selected from the group consisting of: antibiotic resistance, fluorescence, tetracycline-inductance, and a combination of these. 
     
     
         8 . The method of  claim 1 , wherein the recombination vector encodes two or more different selectable markers, wherein the nucleic acid sequence encoding each selectable marker is operably linked to the test nucleic acid sequence. 
     
     
         9 . The method of  claim 8 , wherein at least one of the selectable markers is antibiotic resistance. 
     
     
         10 . The method of  claim 8 , wherein at least one of the selectable markers is fluorescence. 
     
     
         11 . The method of  claim 8 , wherein the two or more selectable markers comprise a nucleic acid sequence encoding a kanamycin resistance gene and a nucleic acid sequence encoding a spectinomycin resistance gene. 
     
     
         12 . The method of  claim 1 , wherein the plurality of phenotypically homogenous prokaryotic cells comprises a plurality of genetically identical prokaryotic cells. 
     
     
         13 . The method of  claim 1 , wherein recombination cloning methods are used to generate the library of recombination vectors. 
     
     
         14 . A library of transformed prokaryotic cells comprising:
 a plurality of prokaryotic cells of the same species and strain, each prokaryotic cell comprising a recombination vector, wherein a majority of the prokaryotic cells have a different recombination vector than the other cells in the library, wherein each recombination vector comprises:
 a test nucleic acid sequence encoding a candidate biologically active random peptide (BARP), wherein each test nucleic acid sequence consists of nucleic acids encoding, in the following order: a start codon, a spacer codon selected from alanine or glycine, a first cystine residue, a random sequence of 6-20 amino acids representing a candidate BARP, a second cysteine residue, such that the first and second cysteine residues flank the random sequence of amino acids, and a stop codon, and wherein the test nucleic acid sequence is flanked by recombinatorial cloning primer sequences; and 
 a nucleic acid sequence encoding a selectable marker operably linked to the test nucleic acid sequence, 
   
       wherein the test nucleic acid sequence in a plurality of the vectors encodes a different random sequence of amino acids from the other vectors and wherein the plurality of prokaryotic cells is phenotypically homogeneous in the absence of the recombination vector. 
     
     
         15 . The library of  claim 14 , wherein the random sequence of amino acids is 6 amino acids in length and wherein each test nucleic acid in the library consists of SEQ ID NO: 1, wherein “n” represents any nucleotide, and wherein each “n” for each test nucleic acid sequence in the library is independently selected. 
     
     
         16 . The library of  claim 14 , wherein the random sequence of amino acids is 12 amino acids in length. 
     
     
         17 . The library of  claim 14 , wherein the selectable marker is selected from the group consisting of antibiotic resistance, fluorescence, tetracycline-inductance, and a combination of these. 
     
     
         18 . The library of  claim 14 , wherein the recombination vector encodes two or more different selectable markers, wherein the nucleic acid sequence encoding each selectable marker is operably linked to the test nucleic acid sequence. 
     
     
         19 . The library of  claim 18 , wherein at least one of the selectable markers is antibiotic resistance. 
     
     
         20 . The library of  claim 14 , wherein the prokaryotic cells are bacterial cells.