326 FliTrx bacterial display library (X12) 12 1.77e8 2.0e10 Invitrogen, Carlsbad, CA Completely random Linear The FliTrx(TM) Random Peptide Display Library is an E. coli-based system that eliminates the use of phage and makes screening of peptide interactions faster and easier. The FliTrx(TM) Library was constructed in the pFliTrx(TM) vector. A diverse library of random dodecapeptides is positioned in the active site loop of the thioredoxin protein (trxA), inside the dispensable region of the bacterial flagellin gene (fliC). The resultant recombinant fusion protein (FLITRX) is exported and assembled into partially functional flagella on the bacterial cell surface. The FliTrx library, which expresses random dodecapeptides flanked by cysteine-glycine-proline/glycine-proline-cysteine sequences (CGP/GPC) on the cell surface as FliC-Trx fusions. 327 OmpX7C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 4.0e9 Patrick S. Daugherty Semi-random NNS Circular Peptide insertion library within OmpX of the form X(2)-C-X(7)-C-X(2) was constructed using PCR with primers PD671/PD516, using as a template pB33OmpXtemp. The resulting products were digested with SfiI and ligated into SfiI-digested pB33OmpXT2. Ligation products were desalted and electroporated into electrocompetent MC1061 yielding 4.0e9, For CPX library construction, a PCR template was created (pB33CPX-template) lacking a passenger peptide and incorporating a silent mutation that destroys the SfiI restriction site. 328 OmpX3C bacterial display library ( X(4)-C-X(3)-C-X(4) ) 13 1.3e9 Patrick S. Daugherty Semi-random NNS Circular Peptide insertion library within OmpX of the form X(4)-C-X(3)-C-X(4) was constructed using PCR with primers PD671/PD516, using as a template pB33OmpXtemp. The resulting products were digested with SfiI and ligated into SfiI-digested pB33OmpXT2. Ligation products were desalted and electroporated into electrocompetent MC1061 yielding 1.3e9, For CPX library construction, a PCR template was created (pB33CPX-template) lacking a passenger peptide and incorporating a silent mutation that destroys the SfiI restriction site. 329 CX7(1)C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 4.0e8 Patrick S. Daugherty Semi-random NNS Circular CX7(1)C bacterial display library was generated in a CPX vectors. PCR was performed with primers PD707 and PD634 using pB33CPX-template. The resulting PCR product was lengthened in a second PCR to enable efficient digestion, using primers PD180 and PD753. This product was digested with SfiI and ligated into HincII/SfiI digested pB33CPX-template. 330 CX7(2)C bacterial display library ( X(2)-C-X(7)-C-X(2) ) 13 1.0e10 Patrick S. Daugherty Semi-random NNS Circular CX7(2)C bacterial display library was constructed using a CPX scaffold containing the substitutions L(-17)V, L(-14)V, L(-10)V, L26V, L37I, L113V, L123V, where the residue numbering is based on the wild-type OmpX. A plasmid encoding a CPX without leucine codons (pB33NLCPX) was constructed by overlap PCR. PCR products generated with primers PD515/PD703, PD704/PD632, and PD633/PD634 were used for overlap PCR with outside primers PD515/PD634. The resulting product was cloned into KpnI/HindIII digested pBAD33. The CX7(2)C bacterial display library was then constructed by PCR amplification of pB33NLCPX with primers PD707/PD180, amplification of the product with PD753/PD180, digestion with SfiI, and ligation into SfiI-digested pB33NLCPX. 331 X15 CPX bacterial display library 15 Patrick S. Daugherty Completely random NNS Linear X15 CPX bacterial display library was constructed as fusions to the extracellular N-terminus of a circularly permuted variant of outer membrane protein OmpX (CPX). Following the native signaling sequence of OmpX, CPX was constructed by fusion of the native C- and N-terminus of OmpX with a GGSG linker, and by opening loop 2 between residues S53 and S54 yielding a scaffold with extracellular C- and N-termini. 332 CX9C E. coli bacterial display library 9 Sang J. Chung Completely random NNN Linear The FcBP1-eGFP vector, which encodes a fusion protein of eGFP with FcBP1, at the C-terminus of FcBP1, was newly designed and constructed by PCR. An FcBP-eGFP DNA library was constructed by inserting 27 nt-randomized DNA sequences into the N-terminus of the eGFP gene by PCR using the eGFP gene as template under the conditions described above (50-primer: 50-GCC GCA GCC ATA TGA AAG ATG AAT GTN NNN NNN NNN NNN NNN NNN NNN NNN NNT GCG AAT TCG AGC TCC GTC GA-30; 30-primer, ATT CGC GGA TCC CTT GTA CAG CTC GTC CAT GCC GAG; where N is A, T, G, or C, and the restriction sites for NdeI and BamHI are italicized). The purified PCR product was digested with NdeI and BamHI and cloned into pHCE-IIB, yielding the FcBP-eGFP DNA library. 333 PL12 yeast display library (X12) 12 Angela M. Belcher Completely random NNK Linear A yeast display peptide library was created from a degenerate oligonucleotide pool encoding random 12-mer peptides with a NNK bias. Peptides were displayed as fusions to the C-terminus of Aga2, which is encoded on a plasmid downstream of a Gal-based promoter. The expression vectors were maintained in S. cerevisiae strain EBY100, which has Aga1 under control of a Gal-based promoter integrated in its genome, Oligonucleotides used in library construction were obtained from Oligos, Etc. The degenerate oligonucleotide pool encoding the random 12-mer peptide library (PL12LibrBstXI) 50-pGTGGCGGTAGCGGC-(NNK)12-TAGCTAGCTAGGCCAGTAGC, synthesized with an NNK bias in order to reduce the stop codon frequency, was annealed to two short primers (5'BstXIanneal) 5'-pGCCGCTACCGCCACCGCC and (3'BstXIanneal) 5'-pCTGGCCTAGCTAGCTA. The resulting hybrid DNA library containing duplexed BstXI sticky ends was directly ligated into the BstXI sites of pBPZ, and electroporated into E. coli Electromax DH10B (Invitrogen, Carlsbad, CA) for amplification on plated LB Ampt media. 334 LAP yeast display library 12 Alice Y. Ting Semi-random Linear The LAP library is displayed on the yeast surface as a fusion to Aga2p protein. A C-terminal myc epitope is used to quantify LAP expression level. A partially randomized oligo with the following sequence: 5'A AAT AAG CTT TTG TTC GGA TCC NGM MNN NAN NTS MNN MNN AAC TTT ATC MNN NTS NAN TCC GCT AGC CGA CCC TCC, was ordered from IDT (Integrated DNA technologies). N designates an equimolar mixture of all bases. S designates a 1:1 mixture of G and C. M designates a 1:1 mixture of A and C. This oligo was annealed with another oligo, Con2For.F (5'CT AGT GGT GGA GGA GGC TCT GGT GGA GGC GGT AGC GGA GGC GGA GGG TCG GCT AGC GGA), which overlaps with both pCTCON2 vector and the library oligo. The 5' overhangs were filled in using Klenow polymerase. The resulting product was PCR-amplified using the primers Con2For.F and Con2Rev.R (5'TA TCA GAT CTC GAG CTA TTA CAAGTC CTC TTC AGA AAT AAG CTT TTG TTC GGA TCC). Meanwhile, pCTCON2 vector was prepared by digestion with NheI and BamHI, and gel-purified. PCR insert and pCTCON2 vector were transformed together into S. cerevisiae EBY100 (Invitrogen) by electroporation as described by Colby et al.52 Homologous recombination occurred inside the yeast. Serial dilutions of transformed yeast were plated on SDCAA plates and colonies were counted, to determine transformation efficiency. 335 SC-X(16)-C M13 phage display library 16 Mark W. Grinstaff Completely random NNK Circular 336 X8 M13 phage display library 8 Angela M. Belcher Completely random NNM Linear The M13KE phage vector was modified by making a cloning site for pVIII display. A Pst I restriction site was made by mutating T to A at position 1372, a BamH I site was made by mutating C to G at positon 1381, and the Pst I site at position 6246 was deleted by mutating T to A at position 6250. The site-directed mutagenesis was done using overlap extension PCR. A dsDNA library was then prepared and cloned into the resulting modified phage vector, named M13SK, using Pst I and BamH I. To obtain the dsDNA library, partial library duplexes were formed by annealing of extension primer (5\'-GATGCTGTCTTTCGCTGCAG-3\') with oligonucleotides (3\'-ACGACAGAAAGCGACGTCnm(nnm)6nnCCTAGGAACATC ATC-5\', where n=A, T, C, or G and m=A or C). The partial library duplexes were incubated with Klenow fragment (3\'→'5\' exo-) (10 U/μL) and dNTP at 37 °C for 30 min. The Klenow fragment was inactivated by heating (75 °C for 20 min), and the mixture was digested with Pst I and BamH I. The digested DNA was gel purified (2-40% TBE polyacrylamide gel), ligated into M13SK, and transfected to XL1-Blue Electroporation Competent Cells using a MicroPulser (Biorad). The library was titered according to manufacturer directions and sequenced (MIT Biopolymers Laboratory) before amplification. 337 X9 fUSE5 phage display library 9 Po Tien Completely random NNK Linear The plasmid was constructed in such a way that only clones bearing frame-restoring inserts could form infectious particles in this library. A large number of random 27-mers were synthesized and digested by BglI before they were inserted near the amino terminus of the minor coat-protein gene (gIII) of fUSE5. A phage particle could only express one of the random 27-mer sequences on its surface as a fusion protein of pIII. 338 X6 fUSE5 phage dislpay library 6 8.6e8 2.5e13 Ivone M. Takenaka Completely random NNM Linear The 6-mer phage display random peptide library was constructed using the fUSE-5 vector, kindly provided by G. Smith (University of Missouri). FUSE-5 replicative Form DNA was purified from K802 Escherichia coli and linearized by SfiI digestion, and the resulting vector DNA was ligated to synthetic DNA fragments containing complementary BglI termini. These duplex oligonucleotides contained (NNM)6 triplets in the coding strand, where N corresponds to equivalent amounts of A, T, G, and C and where M corresponds to equivalent amounts of G and T (minimizes occurrence of translational stop codons). The noncoding strand consisted of 18 or 45 residue stretches of the inosine nucleotide analog. Recombinant DNA was electroporated into MC1061 E. coli electrocompetent cells, spread on 94 plates (24 x 24 cm) with NZamine-yeast extract (NZY) agar plus 40 μg/ml tetracycline (tet) and incubated overnight at 37 °C. Phage were collected by polyethylene glycol precipitation and purified on cesium chloride gradients. The number of transducing units (TU) was determined by infecting K91Kan-resistant cells with an aliquot from each library and plating on tet plates. FUSE5-vector DNA containing an insert restores the reading frame of the pIII gene product, enabling the phage to infect K91Kan-resistant cells and produce colonies on tet plates. 339 TN6-6 phage display library 12 Dyax Corp Semi-random The disulfide-constrained cyclic peptide phage display library TN6-6 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN6-6 phage library is depicted as "AEGTGS-X1X2X2 CX2X2X2X2C X2X2X1-APGPTDS", where X1=ADFGHLNPQRSVWY and X2=ADEFGHIKLMNPQRSTVWY. 340 TN12-1 phage display library 18 Dyax Corp Semi-random Circular The disulfide-constrained cyclic peptide phage display library TN12-1 was constructed in a derivative of M13mp18, MANP, having the following modifications: bla (AmpR) gene, modified junction between signal of iii and coding region for mature III, and removal of LacZ complementation system. MANP comprises 8164 bases. The bla gene was from pGEM3Zf1(+), was bound by BamHI-HindIII sites at the 5' end and HindIII-SalI sites at the 3' end, replaced bases 6001 through 6432 of M13mp18, and was in the same orientation as the phage genes. The junction between the III signal sequence and mature III was modified with an NcoI restriction site embedded in the last three codons of the signal sequence. After the III signal, MANP contained the sequence of BPTI with restriction sites for the enzymes StyI, XhoI, PflMI, ApaI, Bsp120I, EcoO109I, PspOMI, BssHII, StuI, BstZ17I, BlpI, EagI, and SphI, which were all unique within MANP. Following BPTI, a unique PstI site and a Factor Xa cleavage site were inserted in a short linker region before mature III. The DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using TAQ DNA polymerase, cleaved with NcoI and PstI, and ligated to similarly cleaved vector. The variegated parts were synthesized with TRIM technology, which incorporates trinucleotides and allows mixtures of any set of amino acid types in any desired proportions. The flanking and varied DNA sequence positions of TN6-6 phage library is depicted as "AEGTGD-X1X1X2 CX2X2X2X2X2X2X2X2X2X2C X2X1X1-APGPTDN", where X1=ADFGHLNPRSWY and X2=ADEFGHIKLMNPQRSTVWY. 341 fUSE5-based X10 phage display library 10 2.0e7 B. H. Lindqvist Completely random NNK Linear 342 XCX8CX+X15+X8CX8+X30 M13 phage display library pool 12, 15, 17, 30 Claude Granier Semi-random NNK Linear 343 CX7C T7 phage display library 7 In-San Kim Completely random Circular 344 CX8C phage display library 8 Kristiina Aalto Completely random Circular 345 X3 T7 phage display library 3 Shunsuke Kamijo Completely random Linear The phage display library is based on T7 415-1b phage vector. 346 X4 T7 phage display library 4 Shunsuke Kamijo Completely random Linear The phage display library is based on T7 415-1b phage vector. 347 fUSE5 phage display library Erkki Ruoslahti Semi-random 348 X10-ALLRY-X10 phage display library 25 Igor Fisch Semi-random NNK Linear Illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage fd by fusion to the pIII phage coat protein and selected by binding to several proteins, including β-glucuronidase. 349 X6 fdMED1 phage display library 6 Jean-Pierre Mach Completely random NNK Linear The vector fdMED1 was used for the library construction. In order to avoid the background production of wild-type M13 phage during the selections, a new polylinker cloning site containing several stop codons was constructed between the two unique restriction sites ApaLI and NotI of the original fdDOG1 vector. The construction of this new vector (fdMED1) was performed as follows: the peptide leader sequence (LpelB) of the vector backbone pHEN1 was amplified by PCR with Taq polymerase (Perkin Elmer) using oligo-1 and oligo-fdSEQ01FOR. The reaction mixture (50 mL) was cycled 30 times. The resulting DNA fragment was subcloned into ApaLI and NotI digested fdDOG1 and used to transform Escherichia coli TG1. Clones containing the correct insert were identified by DNA sequencing. In addition, a multiple cloning site (MCS) was generated by inserting annealed oligonucleotides (oligo-4 and oligo-5) into SfiI/NotI digested vector. The DNA was electroporated in E. coli MC1061, thus generating a new fd vector called fdMED1. 350 CX6C fdMED1 phage display library 6 Jean-Pierre Mach Completely random NNK Circular The vector fdMED1 was used for the library construction. In order to avoid the background production of wild-type M13 phage during the selections, a new polylinker cloning site containing several stop codons was constructed between the two unique restriction sites ApaLI and NotI of the original fdDOG1 vector. The construction of this new vector (fdMED1) was performed as follows: the peptide leader sequence (LpelB) of the vector backbone pHEN1 was amplified by PCR with Taq polymerase (Perkin Elmer) using oligo-1 and oligo-fdSEQ01FOR. The reaction mixture (50 mL) was cycled 30 times. The resulting DNA fragment was subcloned into ApaLI and NotI digested fdDOG1 and used to transform Escherichia coli TG1. Clones containing the correct insert were identified by DNA sequencing. In addition, a multiple cloning site (MCS) was generated by inserting annealed oligonucleotides (oligo-4 and oligo-5) into SfiI/NotI digested vector. The DNA was electroporated in E. coli MC1061, thus generating a new fd vector called fdMED1.