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 nucleic acid fragments derived from one or more microorganisms or eukaryotes containing compact genomes, each of said microorganisms or eukaryotes having a substantially sequenced genome, wherein the nucleic acid fragments of the library comprise an open reading frame having an average length of at least about 36-45 nucleotide residues and/or encode a protein domain, and wherein the nucleic acid fragments are inserted into an expression vector thereby producing recombinant constructs wherein each fragment is in operable connection with a promoter sequence that is capable of conferring expression of that fragment. 
     
     
         92 . An expression library comprising nucleic acid fragments derived from one or more microorganisms or eukaryotes containing compact genomes, each of said microorganisms or eukaryotes having a substantially sequenced genome, wherein the nucleic acid fragments of the library have substantially different nucleotide sequences and wherein the nucleic acid fragments are inserted into an expression vector thereby producing recombinant constructs wherein each fragment is in operable connection with a promoter sequence that is capable of conferring expression of that fragment. 
     
     
         93 . The expression library of  claim 91  wherein the nucleic acid fragments are derived from an organism 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;    
     
     
         94 . The expression library of  claim 92  wherein the nucleic acid fragments are derived from an organism 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 expression vector is a plasmid, bacteriophage or phagemid. 
     
     
         96  The expression library of  claim 95  wherein the expression vector comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 60 and SEQ ID NO: 61. 
     
     
         97 . The expression library of  claim 92  wherein the expression vector is a plasmid, bacteriophage or phagemid. 
     
     
         98 . The expression library of  claim 97  wherein the expression vector comprises the nucleotide sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 60 and SEQ ID NO: 61. 
     
     
         99 . The expression library of  claim 91  or  92  wherein the number of genomes of a microorganism or eukaryote that is represented in the library is more than one. 
     
     
         100 . The expression library of  claim 99  wherein the nucleic acid fragments are represented in an amount proportional to the size of the genome of a microorganism or eukaryote from which the fragments were derived. 
     
     
         101 . The expression library of  claim 91  or  92  wherein the recombinant vector is in a cellular host. 
     
     
         102 . The expression library of  claim 101  wherein the cellular host is a bacterium. 
     
     
         103 . The expression library of  claim 101  wherein the cellular host is a yeast. 
     
     
         104 . The expression library of  claim 101  wherein the cellular host is a nematode. 
     
     
         105 . The expression library of  claim 104  wherein the nematode is  C. elegans.    
     
     
         106 . A database comprising the nucleotide sequences of nucleic acid fragments of the expression library of  claim 91  or the amino acid sequences of the peptides encoded by said nucleic acid fragments in computer readable form. 
     
     
         107 . A database comprising the nucleotide sequences of nucleic acid fragments of the expression library of  claim 92  or the amino acid sequences of the peptides encoded by said nucleic acid fragments in computer readable form.