Methods of Constructing and Screening Diverse Expression Libraries

Abstract

The present invention provides novel methods for producing nucleic acid fragment libraries that express highly diverse peptides or protein domains and, in particular, methods for producing nucleic acid fragment libraries wherein the nucleic acid fragments of the libraries are derived from two or more diverse characterized genomes.

1 - 90 . (canceled) 
     
     
         91 . An expression library comprising a plurality of recombinant constructs, wherein each construct comprises
 a nucleic acid of up to 250 nucleotides in length, wherein the nucleic acid is identical to a fragment of a genomic region of an organism having a compact genome for which at least 60% of the genome has been characterised by sequencing, and   a promoter operably-linked to the nucleic acid;   
       wherein the expression library comprises nucleic acids identical to fragments of genomic regions of at least two organisms and wherein the nucleic acids are present in the expression library in an amount proportional to the size of the organism's genome to which the nucleic acids are identical such that the expression library has about equal representation of each organism's genome. 
     
     
         92 . The expression library of  claim 91 , wherein the nucleic acid is an amplification product obtained by amplifying genomic DNA using random oligonucleotide primers and wherein the nucleic acid has been inserted separately by ligation reaction performed in vitro into a recombinant gene construct. 
     
     
         93 . The expression library of  claim 91 , wherein the expression library comprises open reading frames having a length of at least 36 nucleotide residues, wherein the open reading frames encode peptides capable of binding to a protein or nucleic acid in a cell. 
     
     
         94 . The expression library of  claim 91 , wherein the nucleic acid are from organisms selected from the group consisting of:  Aeropyrum pernix, Anopheles gambiae, Arabidopsis thaliana, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Caenorhabditis elegans, Chlamydia trachomatis, Danio rerio, Drosophila melanogaster, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Saccharomyces cerevesiae, Schizosaccharomyces pombe, Synechocystis  PCC 6803 , Takifugu rubripes, Thermoplasma volcanium , and  Thermotoga maritima.   
     
     
         95 . The expression library of  claim 91 , wherein the nucleic acid are from organisms selected from the group consisting of:  Aeropyrum pernix, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, Danio rerio, Drosophila melanogaster, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Synechocystis  PCC 6803 , Thermoplasma volcanium , and  Thermotoga maritima.   
     
     
         96 . The expression library of  claim 91 , wherein the nucleic acid are from:  Aeropyrum pernix, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Synechocystis  PCC 6803 , Thermoplasma volcanium , and  Thermotoga maritima.   
     
     
         97 . The expression library of  claim 91 , wherein each recombinant construct is a plasmid vector, bacteriophage vector or phagemid vector. 
     
     
         98 . The expression library of  claim 91 , wherein each recombinant construct is a bacteriophage vector or plasmid vector comprising SEQ ID NO: 36. 
     
     
         99 . The expression library of  claim 91 , wherein each recombinant construct is a bacteriophage vector and wherein the expression library comprises recombinant bacteriophage particles. 
     
     
         100 . The expression library of  claim 99 , wherein the bacteriophage vector is a M13 bacteriophage vector. 
     
     
         101 . The expression library of  claim 99 , wherein the bacteriophage vector is a T7 bacteriophage vector. 
     
     
         102 . The expression library of  claim 91 , wherein no two organisms share more than 90% sequence identity at the nucliec acid level.