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 isolated nucleic acid fragments from nineteen organisms selected from microorganisms and eukaryotes containing compact genomes that are substantially sequenced, wherein:
 (i) the isolated nucleic acid fragments from each of said organisms are present in said expression library in amounts that are proportional to the relative genome sizes of said organisms;   (ii) the isolated nucleic acid fragments comprise fragments of known nucleotide sequences that are predicted to encode protein domains of polypeptides of known function;   (iii) the isolated nucleic acid fragments comprise open reading frames having an average length of 36-45 nucleotide residues, wherein the open reading frames comprises open reading frames encoding peptides having activities not predictable from the known function; and   (iv) the isolated nucleic acid fragments are inserted separately into an expression vector thereby producing recombinant constructs wherein each of said fragments is in operable connection with a promoter sequence that confers expression of that fragment.   
     
     
         92 . The expression library of  claim 91  wherein the isolated nucleic acid fragments 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.    
     
     
         93 . The expression library of  claim 91  wherein the isolated nucleic acid fragments 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.    
     
     
         94 . The expression library of  claim 91  wherein the isolated nucleic acid fragments 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.    
     
     
         95 . The expression library of  claim 91  wherein the expression vector is a plasmid vector, bacteriophage vector or phagemid vector. 
     
     
         96 . The expression library of  claim 91  wherein the expression vector is a bacteriophage vector plasmid vector comprising SEQ ID NO: 36. 
     
     
         97 . The expression library of  claim 91  wherein the recombinant construct comprises a vector in a cellular host. 
     
     
         98 . The expression library of  claim 97  wherein the cellular host is a bacterium. 
     
     
         99 . The expression library of  claim 97  wherein the cellular host is a yeast. 
     
     
         100 . The expression library of  claim 91  wherein the expression vector is a bacteriophage vector and wherein the expression library comprises recombinant bacteriophage particles. 
     
     
         101 . The expression library of  claim 100 , wherein the bacteriophage vector is M13. 
     
     
         102 . The expression library of  claim 100 , wherein the bacteriophage vector is a T7 bacteriophage vector. 
     
     
         103 . A bacteriophage expression library comprising isolated nucleic acid fragments comprising open reading frames each having an average length of 36-45 nucleotide residues, said fragment being from the organisms  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 , and wherein the fragments are inserted separately into a bacteriophage expression vector thereby producing recombinant constructs wherein each of said fragments is in operable connection with a promoter sequence that confers expression of that fragment. 
     
     
         104 . The bacteriophage expression library of  claim 103 , wherein the bacteriophage vector is M13. 
     
     
         105 . The bacteriophage expression library of  claim 103 , wherein the bacteriophage vector is a T7 bacteriophage vector.