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Electronic Supplementary Material for

Cox et al. (2007) Protein fabrication automation Protein Sci. 16(3)

Description of ESM included in this file:

1) Annotated sequence maps of the pSOS, pSOX, and pSOS-X vectors

2) A spreadsheet listing the genetic elements of the above vectors

3) Several tables indicating the outcome of our various optimization efforts

4) Expanded Materials and Methods covering those optimizations and other

routine molecular biology steps

Electronic supplementary materials for “Protein Fabrication Automation” in Protein Science, by

Cox et al.

Provided below is the vector sequence information for pSOS (Genbank #EF030424). FEATURES Location/Qualifiers CDS 1676..2469 /note="aphA1 gene, Aminoglycoside 3'-phosphotransferase enzyme, E.C. 2.7.1.95" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" CDS 116..772 /note="cat gene, chloramphenicol acetyletransferase enzyme, E.C. 2.3.1.28" /translation="EKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS SLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVA NMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA " protein_bind 76..83 /note="PacI restriction site" protein_bind 92..97 /note="HpaI restriction site" promoter 1538..1675 /note="aphA1 promoter" rep_origin 3163..3836 /note="pMB1 plasmid replication origin" RBS 70..75 /note="cloneing site Shine Delgarno sequence" promoter 4053..4174 /note="Lac promoter"

^^ ctatagaatactcaagctatgcatcaagctctagtaacggccgccagtgtgctggaattcgcccttgttaaggagttaattaagactgctagttaacggcggtagcggtggctcggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttcagtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatgaaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgaggagggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctatttaggtgaca

Electronic supplementary materials for “Protein Fabrication Automation” in Protein Science, by

Cox et al.

Provided below is the vector sequence information for pSOX (Genbank #EF030425). FEATURES Location/Qualifiers rep_origin complement(828..240) /note="pMB1 plasmid replication origin" CDS complement(1859..999) /note="bla gene, beta lactamase enzyme, E.C. 3.5.2.6" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" RBS complement(1871..1867) /note="bla RBS" promoter complement(1903..1929) /note="bla promoter" promoter 2106..2151 /note="lacI promoter" RBS 2160..2166 /note="lacI RBS" CDS 2172..3254 /note="lac I, lactose operon repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" promoter 3503..3530 /note="Ptac promoter" RBS complement(3611..3616) /note="cloning site Shine Delgarno sequence" protein_bind 3617..3624 /note="PacI restriction site" protein_bind 3633..3640 /note="PmeI restriction site" terminator 3813..4016 /note="rrnB T1T2 TXN terminator"

^^ CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGATAAGCTTCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACAATTCTCATGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGCTCGAGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCCAGTCCGTTTAGGTGTTTTCACGAGCCATTCACCAACAAGGAGTTAATTAACTATCATAGTTTAAACTAACTAGTGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCG

Electronic supplementary materials for “Protein Fabrication Automation” in Protein Science, by

Cox et al.

Provided below is the vector sequence information for pSOS-X (Genbank #EF030426). FEATURES Location/Qualifiers rep_origin complement(828..240) /note="pMB1 plasmid replication origin" CDS complement(1859..999) /note="bla gene, beta lactamase enzyme, E.C. 3.5.2.6" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA EIGASLIKHW" RBS complement(1871..1867) /note="bla RBS" promoter complement(1903..1929) /note="bla promoter" promoter 2106..2151 /note="lacI promoter" CDS 2172..3254 /note="lac I, lactose operon repressor" /translation="VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT QTASPRALADSLMQLARQVSRLESGQ" terminator 4495..4698 /note="rrnB T1T2 TXN terminator" promoter 3503..3530 /note="Ptac promoter" RBS complement(3611..3616) /note="cloning site Shine Delgarno sequence" protein_bind 3617..3624 /note="PacI restriction site" misc_feature 3639..3656 /note="polyhistidine protein purification tag" misc_feature 3657..3659 /note="amber stop codon" misc_feature 3633..3638 /note="NaeI restriction site" CDS 3666..4319 /note="cat gene, chloramphenicol acetyltransferase enzyme, E.C. 2.3.1.28" /translation="EKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFS SLWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVA NMDNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA "

^^ CATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGATAAGCTTCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACAATTCTCATGTTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGCTCGAGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCCAGTCCGTTTAGGTGTTTTCACGAGCCATTCACCAACAAGGAGTTAATTAACTATCATAGCCGGCCACCATCACCATCACCATTAGACTAGTGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATGACTAGTGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAA

CTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCG

Electronic supplementary materials for “Protein Fabrication Automation”

in Protein Science, by Cox et al.

Vector Element Source Genbank Start End

pSOS pMB1 ori pUC19c L09137 1455 867

Tn 903 KanR pACYC177 X06402 2798 3025

Lac promoter pUC19c L09137 543 470

Tn 9 CamR pACYC184 X06403 3805 219

pSOX pMB1 ori pUC19c L09137 1455 867

Tn 3 AmpR pBR322 J01749 4153 3293

pTac promoter pMal-pIII AF031088 1406 1433

rrnB T1T2 pMal-pIII AF031088 3161 3364

Lac Iq pMal-pIII AF031088 81 1163

pSOS-X pMB1 ori pUC19c L09137 1455 867

Tn 3 AmpR pBR322 J01749 4153 3293

pTac promoter pMal-pIII AF031088 1406 1433

rrnB T1T2 pMal-pIII AF031088 3161 3364

Lac Iq pMal-pIII AF031088 81 1163

Tn 9 CamR pACYC184 X06403 3805 219

Electronic supplementary materials for “Protein Fabrication Automation” in Protein

Science, by Cox et al.

Outcomes of various optimization trials are provided on the following pages.

Plus and minus symbols are used to denote the outcome, as described underneath each set

of experiments. References are provided when available.

Polymerase system performance in fragment assembly ..................................................... 2

Effect of oligonucleotide length on fragment assembly ..................................................... 3

Effect of number of oligonucleotide pairings on fragment assembly................................. 4

Oligonucleotide concentration constant (c) performance upon fragment assembly........... 5

Total oligonucleotide concentration studies in fragment assembly.................................... 6

The effect of annealing temperature in fragment assembly reactions ................................ 7

Thermalcycling dependence in fragment assembly............................................................ 8

Effect of additives on PCR assembly reactions .................................................................. 9

References......................................................................................................................... 10

Polymerase system performance in fragment assembly

Polymerase (source) Reference Effect

KOD Hot-Start DNA polymerase

(EMD Biosciences) [1, 2] +++

Taq DNA polymerase

(Invitrogen) +

Cloned Pfu DNA polymerase

(Stratagene) [3-5] +

Accuprime Pfx DNA polymerase

(Invitrogen) ++

Platinum Pfx DNA polymerase

(Invitrogen) +

Tgo DNA polymerase

(Roche Applied Science) -

Expand High Fidelity DNA polymerase

(Roche Applied Science) [6] +

+++ Produced band of correct mass in all initial test conditions

++ Produced band of correct mass in some initial test conditions

+ Produced smears in initial test conditions

- Failed to produce any amplification products in initial test conditions

2

Effect of oligonucleotide length on fragment assembly

Oligonucleotide length Reference Effect

35-mers +

40-mers [1, 4] +

50-mers [7] +

60-mers [2, 8] +

+ reliably yielded assembly products

3

Effect of number of oligonucleotide pairings on fragment assembly

Number of oligonucleotide pairings Reference Effect

2 pairs ++

3 pairs ++

4 pairs ++

5 pairs [2] ++

6 pairs [2] +

10 pairs -

13 pairs -

++ Robust for ION-PCR (fragments assemble >99%)

+ Sometimes robust for ION-PCR (fragments assemble >85%)

- Fragments did not assemble into a band of appropriate mass

4

Oligonucleotide concentration constant (c) performance upon fragment assembly

Oligonucleotide concentration constant Reference Effect

c = 0.40 -

c = 0.50 +

c = 0.65 ++

c = 0.70 ++

c = 0.75 ++

c = 0.80 +

c = 0.85 +

c = 1.0 +

++ Yielded bands of correct mass

+ Yielded smears that cover corrent mass

- Yielded smear of incorrect mass

5

Total oligonucleotide concentration studies in fragment assembly

Total oligonucleotide concentration / fragment (nM) Reference Effect

150 -

200 +

250 +

300 +

350 +

400 +

450 +

500 [2] ++

550 ++

600 [2] ++

650 ++/-

700 ++/-

750 ++/-

++ Yielded a strong band of correct mass

+ Yielded a weak band of correct mass

- Failed to yield product

++/- Yielded a strong band of correct mass with higher molecular weight smears

6

The effect of annealing temperature in fragment assembly reactions

Annealing temperature of assembly reaction (ºC) Reference Effect

50.0 +

50.5 +

51.7 +

53.2 +

55.0 +

55.5 +

58.0 +

58.4 ++

60.0 [2] ++

60.6 ++

61.2 ++

61.8 ++

62.4 ++

64.6 ++

66.8 +

68.4 -

69.6 -

70.0 -

++ Produced band of correct mass in all tested assemblies

+ Produced band of correct mass in some tested assemblies

- Failed to produce product

7

Thermalcycling dependence in fragment assembly

Number of ION-PCR thermal-cycles Reference Effect

12 +

14 ++

15 ++

16 ++

18 ++

20 ++

25 ++

30 +/-

32 +/-

33 +/-

35 +/-

++ Yielded strong band of correct mass

+ Yielded faint band of correct mass

+/- Yielded strong band of correct mass, sometimes with higher molecular weight

products.

8

Effect of additives on PCR assembly reactions

PCR enhancement compounds (amount) Reference Effect

Sulfolane

(0.3M) [9] -

Betaine

(0.5M, 1.0M, 1.3M) [10] -

DMSO

(2%, 3.5%, 5%) [11] -

Tetramethylammonium oxalate

(2mM) [12] --

2-pyrrolidone

(1%) [13] -

E. coli single-strand DNA binding protein

(1µg/50µl reaction) [14] --

Acetamide

(5%) [15] -

Formamide

(3.1%) [16] -

Spermidine

(1mM) [17] -

- Mildly reduced the amount of product produced

-- Prevented product formation

9

References

1. Wu, G., et al., Simplified gene synthesis: A one-step approach to PCR-based gene

construction. J. Biotechnol., 2006. 124(3): p. 496-503.

2. Gao, X., et al., Thermodynamically balanced inside-out (TBIO) PCR-based gene

synthesis: a novel method of primer design for high-fidelity assembly of longer

gene sequences. Nucleic Acids Res., 2003. 31(22): p. e143.

3. Carr, P.A., et al., Protein-mediated error correction for de novo DNA synthesis.

Nucleic Acids Res., 2004. 32(20): p. e162.

4. Stemmer, W.P., et al., Single-step assembly of a gene and entire plasmid from

large numbers of oligodeoxyribonucleotides. Gene, 1995. 164(1): p. 49-53.

5. Hoover, D.M. and J. Lubkowski, DNAWorks: an automated method for designing

oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res., 2002. 30(10):

p. e43.

6. Kodumal, S.J., et al., Total synthesis of long DNA sequences: synthesis of a

contiguous 32-kb polyketide synthase gene cluster. Proc. Natl. Acad. Sci. USA,

2004. 101(44): p. 15573-15578.

7. Young, L. and Q. Dong, Two-step total gene synthesis method. Nucleic Acids

Res., 2004. 32(7): p. e59.

8. Xiong, A.S., et al., A simple, rapid, high-fidelity and cost-effective PCR-based

two-step DNA synthesis method for long gene sequences. Nucleic Acids Res.,

2004. 32(12): p. e98.

9. Chakrabarti, R. and C.E. Schutt, The enhancement of PCR amplification by low

molecular-weight sulfones. Gene, 2001. 274(1-2): p. 293-298.

10. Baskaran, N., et al., Uniform amplification of a mixture of deoxyribonucleic acids

with varying GC content. Genome Res., 1996. 6(7): p. 633-638.

11. Winship, P.R., An improved method for directly sequencing PCR amplified

material using dimethyl sulphoxide. Nucleic Acids Res., 1989. 17(3): p. 1266.

12. Kovarova, M. and P. Draber, New specificity and yield enhancer of polymerase

chain reactions. Nucleic Acids Res., 2000. 28(13): p. E70.

13. Chakrabarti, R. and C.E. Schutt, The enhancement of PCR amplification by low

molecular weight amides. Nucleic Acids Res., 2001. 29(11): p. 2377-2381.

14. Rapley, R., Enhancing PCR amplification and sequencing using DNA-binding

proteins. Mol Biotechnol., 1994. 2(3): p. 295-298.

15. Reysenbach, A.L., et al., Differential amplification of rRNA genes by polymerase

chain reaction. Appl. Environ. Microbiol., 1992. 58(10): p. 3417-3418.

16. Sarkar, G., S. Kapelner, and S.S. Sommer, Formamide can dramatically improve

the specificity of PCR. Nucleic Acids Res., 1990. 18(24): p. 7465.

17. Wan, C.Y. and T.A. Wilkins, Spermidine facilitates PCR amplification of target

DNA. PCR Methods Appl., 1993. 3(3): p. 208-210.

10

Electronic supplementary materials for “Protein Fabrication Automation” in Protein

Science, by Cox et al.

Provided below are supplementary Materials and Methods not provided in the text of

“Protein Fabrication Automation”.

Preparation of an ORF Scaffold ......................................................................................... 2

Gene fragment assembly optimization ................................................................................ 3

Synthetic ORF selection (SOS) cloning .............................................................................. 6

References ........................................................................................................................... 7

Preparation of an ORF Scaffold

A ‘leading’ sequence is added to the 5’ portion of the gene containing a PacI

restriction endonuclease recognition site with five flanking nucleotides. This sequence is

AAGGATTAATTAA, where the PacI site is underlined, the Shine-Delgarno sequence is

italicized, and the bold adenosine denotes the beginning of the start codon. A 3’ ‘tailing’

sequence is added to the end of the gene in the GeneFab scaffold. That sequence is AAC

| GGC GGT AGC | CAC CAT CAC CAT CAC CAT | TGA TAA, where the first codon

is present to satisfy a cloning requirement, the next three codons encode a gly-gly-ser

linker, the next six codons provide a polyhistidine purification tag (Smith et al. 1998),

and the last two codons encode stop signals.

2

Gene fragment assembly optimization

Reaction conditions were explored to maximize robustness of the assembly

reaction. Different polymerase systems were investigated including KOD Hot-Start

DNA polymerase (EMD Biosciences, San Diego, CA), Taq DNA polymerase

(Invitrogen, Carlsbad, CA), Cloned Pfu DNA polymerase (Stratagene, La Jolla, CA),

Accuprime Pfx DNA polymerase (Invitrogen), Platinum Pfx DNA polymerase

(Invitrogen), Tgo DNA polymerase (Roche Applied Science, Indianapolis, IN), and

Expand High Fidelity DNA polymerase (Roche Applied Science).

The behavior of the ION-PCR (inside-out nucleation PCR) variables was

examined in detail. Assembly reactions were assayed at primer lengths ranging from 35-

60 nucleotides. Overlaps used for the ION-PCR assembly reactions were tested between

19-26 bp, as well as overlaps for gene fragment stitching by splice-overlap extension

(SOE, below) at 20-25 bp. To determine the longest ION-PCR fragment length that

could be constructed with high robustness, 2, 3, 4, 5, and 6 pairs of primers were

evaluated. Oligonucleotides were tested with no purification (simple desalting) or with

PAGE purification (Ellington 1997). The concentrations of the ION-PCR

oligonucleotides are chosen according to a geometric progression (see text).

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A range of values for c were tested at values of 0.4, 0.5, 0.65, 0.7, 0.75, 0.8, 0.85 and 1.0.

The total concentrations of primers in an assembly reaction ([P]T)were investigated at

150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, and 750 nM. Temperatures

for the annealing step of the assembly reactions were tested at 50, 50.5, 51.7, 53.2, 55.0,

55.5, 58.0, 58.4, 60.0, 60.6, 61.2, 61.8, 62.4, 64.6, 66.8, 68.4, 69.6, and 70°C. The total

rounds of thermocycling were examined at 12, 14, 15, 16, 18, 20, 25, 30, 32, 33, and 35

cycles. Additives were for evaluated for possible enhancement or increase of robustness

of the assembly reactions. Reagents added to reactions were sulfolane (0.3 M;

(Chakrabarti and Schutt 2001a)), betaine (0.5, 1.0, 1.3 M; (Baskaran et al. 1996)), DMSO

(2, 3.5, 5%; (Winship 1989)), tetramethylammonium oxalate (2 mM; (Kovarova and

Draber 2000)), 2-pyrrolidone (1%; (Chakrabarti and Schutt 2001b)), E. coli single-strand

binding protein (1 µg / 50 µl reaction; (Rapley 1994)), acetamide (5%; (Reysenbach et al.

1992)), formamide (3.1%; (Sarkar et al. 1990)), and spermidine (1 mM; (Wan and

Wilkins 1993)).

T. thermophilus RecA protein had been shown to facilitate incredible

enhancement in multiplex PCR reactions (Shigemori et al. 2005) and so was investigated

in assembly reactions. Thermophilic RecA activity was assayed at 0.2, 0.4, 0.8, 1.6 and

3.2 µg / 50 µl reaction. T. thermophilus RecA was generously supplied by the Oishi

group, and later reconstructed by PFA. RecA was also tested against a 32-fold range of

primer concentrations. Additionally, functionality of RecA was explored in a variety of

PCR buffer conditions between that of the authors’ reported buffer system and the KOD

polymerase buffer system (EMD Biosciences). Buffers were assayed using Tris buffer at

pH 8.0, 8.3, and 8.8, 100 or 500 mM KCl, with and without 60 mM (NH4)2SO4, with and

4

without 1% Triton X-100, and with and without 0.01% BSA (always in the presence of

1.5 mM MgCl2 and 300-400 µM ATP).

5

Synthetic ORF selection (SOS) cloning

The pSOS vector was digested with PacI endonuclease (New England Biolabs,

Ipswich, MA) using standard protocols (Sambrook and Russell 2001). After the

digestion, Antarctic Phosphatase and buffer (New England Biolabs) was added per

manufacturer’s instructions to remove the 5’ phosphoryl group (37°C for 30 min,

inactivated by heat denaturation at 65°C, 5-10 minutes). The vector was then purified on

a QIAquick column (Qiagen, Valencia, CA) and digested with HpaI (New England

Biolabs) without 5’ phosphate removal of this site. The final, double-digested product

was purified by agarose gel extraction, again using a QAIquick column (Qiagen).

Full-length assembly of synthetic ORFs was verified by analytical agarose

electrophoresis. The ORFs were purified using QIAquick PCR clean-up kits (Qiagen).

The 5’ portion of the synthetic ORF was digested with PacI (New England Biolabs) and

is then heat inactivated at 65°C for 20 minutes. The reaction was again purified with a

QIAquick column (Qiagen). Next, a portion of the inactivated reaction and predigested

pSOS-1 vector was added to an equal volume of 2x Ligation Premix (EMD Biosciences),

and the ligation reaction was allowed to proceed at 22°C for one hour. A portion of the

ligation reaction (3 µl of a 10 µl ligation reaction) was used to transform 100 µl of

DH10B competent cells. Transformed cells were grown on LB-agar plates containing 34

µg/ml chloramphenicol (37°C, 36-42 hours) to select for correctly assembled synthetic

ORFs. Plasmid DNA was isolated from chloramphenicol-resistant colonies for sequence

verification and recloning into protein expression vectors.

6

References

Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., and Weissman,

S.M. 1996. Uniform amplification of a mixture of deoxyribonucleic acids with

varying GC content. Genome Res. 6: 633-638.

Chakrabarti, R., and Schutt, C.E. 2001a. The enhancement of PCR amplification by low

molecular-weight sulfones. Gene 274: 293-298.

Chakrabarti, R., and Schutt, C.E. 2001b. The enhancement of PCR amplification by low

molecular weight amides. Nucleic Acids Res. 29: 2377-2381.

Ellington, A.D. 1997. Purification of Oligonucleotides Using Denaturing Polyacrylamide

Gel Electrophoresis. In Short protocols in molecular biology, 3rd ed. (eds. F.

Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K.

Struhl), pp. 2-43 - 42-44. John Wiley & Sons, Inc., New York City.

Kovarova, M., and Draber, P. 2000. New specificity and yield enhancer of polymerase

chain reactions. Nucleic Acids Res. 28: E70.

Rapley, R. 1994. Enhancing PCR amplification and sequencing using DNA-binding

proteins. Mol Biotechnol. 2: 295-298.

Reysenbach, A.L., Giver, L.J., Wickham, G.S., and Pace, N.R. 1992. Differential

amplification of rRNA genes by polymerase chain reaction. Appl. Environ.

Microbiol. 58: 3417-3418.

Sambrook, J., and Russell, D.W. 2001. Molecular cloning : a laboratory manual, 3rd ed.

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Sarkar, G., Kapelner, S., and Sommer, S.S. 1990. Formamide can dramatically improve

the specificity of PCR. Nucleic Acids Res. 18: 7465.

Shigemori, Y., Mikawa, T., Shibata, T., and Oishi, M. 2005. Multiplex PCR: use of heat-

stable Thermus thermophilus RecA protein to minimize non-specific PCR

products. Nucleic Acids Res. 33: e126.

Smith, D.L., Struck, D.K., Scholtz, J.M., and Young, R. 1998. Purification and

biochemical characterization of the lambda holin. J. Bacteriol. 180: 2531-2540.

Wan, C.Y., and Wilkins, T.A. 1993. Spermidine facilitates PCR amplification of target

DNA. PCR Methods Appl. 3: 208-210.

Winship, P.R. 1989. An improved method for directly sequencing PCR amplified

material using dimethyl sulphoxide. Nucleic Acids Res. 17: 1266.

7