ENGINEERING OF ANILINE DIOXYGENASE FOR BIOREMEDIATION AND INDUSTRIAL APPLICATIONS ANG EE LUI NATIONAL UNIVERSITY OF SINGAPORE & UNIVERSITY OF ILLINOIS AT URBANA CHAMPAIGN 2007 ENGINEERING OF ANILINE DIOXYGENASE FOR BIOREMEDIATION AND INDUSTRIAL APPLICATIONS ANG EE LUI B. Eng (Hons.), National University of Singapore A THESIS SUBMITTED FOR THE DEGREE OF PHILOSOPHY IN ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE & UNIVERSITY OF ILLINOIS AT URBANA CHAMPAIGN 2007 Acknowledgements My heartfelt thanks to my advisors, Associate Professor Jeffrey Obbard and Associate Professor Huimin Zhao for the guidance, inspiration, support and patience they have given me throughout my PhD. I am eternally grateful to them for doing everything possible (and more) to help me along this journey. I would like to thank my prelim committee members, Dr Richard Braatz, Dr Nick Sahinidis, and Dr Chris Rao from UIUC, as well as Dr Lanry Yung from NUS, for their advice on my project. I would like to thank my friends in the Zhao lab for all their help in my work, especially Zhilei for her guidance when I first started in the lab. It was hard work but thank you guys so much for making the late nights in the lab so lively and not so lonely! To my friends at UIUC – Mike, Ty, Karina, Nate, Mo, Jon, Zeng Yi, Wenjuan, Karu, Jungkul, Olga, Ryan Woodyer and Ryan Sullivan, Sheryl, Lily, Charlotte, Jing, Kim Seng, Christian, Rob, Neel, Josh, Halong, Alice, Esther, Eng Kiat, the Singapore Students’ Association, the International Football Club, and everyone else – thanks for the great time and making me feel so at home in Urbana Champaign. I would like to thank Jeff’s group for their hospitality and welcoming me into the group as though I was there all along when I returned to Singapore. Also, I never would have finished this work if not for the kindness and generosity of Dr Choe, Haibin and Nian Rui. Thank you very much for putting me up in your lab in NUS. i Special thanks to my mother, Tan Kim Lian, who toiled all these years to give me this opportunity and was always there to give me support. It definitely took a lot of courage and sense of adventure for me to take up this program and I would like to thank my brother, Heng Ung, for inspiring me with both these qualities. To the most special person in my life, Xing Yi, thank you for being beside me through the best and worst times, always giving me advice and more importantly, loving me. Throughout this journey, just like during the Chicago Marathon in 2004, there is no one else I would rather have beside me. I cherish the memories we created together, but look forward even more to our exciting life ahead with you by my side. Lastly, I would like to thank God for always being there for me and giving me strength and hope during difficult times. ii Contents Acknowledgements i Contents iii Summary ix List of Tables xi List of Figures xii Nomenclature xvii Chapter Introduction 1.1 Background and motivation 1.2 Objectives 1.3 References Chapter Literature review 11 2.1 Aromatic amines 11 2.2 Sources of aromatic amines in the environment 12 2.3 Environmental fates of aromatic amines 13 2.4 Toxicity of aromatic amines 14 2.5 Methods of aromatic amine removal 15 2.5.1 Chemical methods 15 2.5.2 Biodegradation of aromatic amines 17 Biomolecular engineering in bioremediation 19 2.6.1 Tools for biomolecular engineering 20 2.6.2 Naphthalene dioxygenase engineering 26 2.6.3 Biphenyl dioxygenase engineering 29 2.6.4 More engineering on dioxygenases 32 2.6 iii 2.7 Conclusion 32 2.8 References 36 Chapter Functional expression of aniline and carbazole dioxygenases 45 3.1 Introduction 45 3.2 Materials and methods 46 3.2.1 Materials 46 3.2.2 pTrcA-2 plasmid construction 47 3.2.3 pTA1-1plasmid construction 47 3.2.4 pTA2-3 plasmid construction 48 3.2.5 Sample preparation for SDS-PAGE analysis 49 3.2.6 Activity assay 49 3.2.7 CarA resting cell assay 50 3.2.8 Identification of carbazole and 2ABPD 51 Cloning of atdA operon into expression vector: pTrcA-2 51 3.3.1 52 3.3 3.4 SDS-PAGE analysis of AtdA expression by pTrcA-2 Introduction of restriction sites flanking AtdA3: pTA2-3 53 3.4.1 54 SDS-PAGE analysis of AtdA expression by pTA2-3 3.5 AtdA activity of various plasmid constructs 56 3.6 Functional expression of carbazole-1,9a-dioxygenase 58 3.7 Preparation of 2’-aminobiphenyl-2,3-diol 60 3.8 Summary 62 3.9 References 63 Chapter Screening and selection methods for AtdA 65 4.1 Introduction 65 4.2 Materials and methods 67 4.2.1 Chemicals 67 4.2.2 Indophenol blue assay 67 iv 4.3 4.4 4.2.3 MBTH assay 67 4.2.4 van Urk reagent assay 68 4.2.5 Gibbs’ reagent assay 68 4.2.6 Gibbs’ reagent solid phase screen 68 4.2.7 Autooxidation screen 69 4.2.8 Selection 69 Screening 70 4.3.1 Indophenol blue 71 4.3.2 MBTH reagent 73 4.3.3 van Urk reagent 75 4.3.4 Gibbs’ reagent assay 77 4.3.5 Solid phase screening 83 4.3.6 Autooxidation 86 Selection 88 4.4.1 Effect of IPTG 88 4.4.2 Minimal ammonium concentration 89 4.5 Summary 93 4.6 References 95 Chapter Substrate specificity of AtdA 97 5.1 Introduction 97 5.2 Materials and methods 98 5.2.1 Materials 98 5.2.2 Substrate specificity assay 99 5.2.3 Construction of plasmids for gene deletion assay 100 5.2.4 Gene deletion studies 101 5.2.5 Whole cell activity assay 102 5.3 Substrate specificity of AtdA 103 5.4 Effect of methyl sidechain position on enzyme activity 107 v 5.5 Gene deletion studies 111 5.6 Summary 115 5.7 References 117 Chapter Probing the molecular determinants of AtdA substrate specificity 119 6.1 Introduction 119 6.2 Materials and methods 121 6.2.1 Materials 121 6.2.2 Homology modeling 122 6.2.3 Saturation mutagenesis 123 6.2.4 Screening method 124 6.2.5 Whole cell activity assay 125 6.2.6 Identification of products 126 6.2.7 Sample preparation for SDS-PAGE analysis 127 6.3 Identification of substrate binding pocket residues 128 6.4 Saturation mutagenesis 130 6.4.1 V205 Library 131 6.4.2 L248 library 132 6.4.3 F348 library 133 6.5 SDS-PAGE analysis 134 6.6 Whole cell activity for 2IPA 136 6.7 Whole cell activity for aniline and 24DMA 136 6.8 Analysis of mutations and discussion on AtdA1 and A2 139 6.9 Summary 144 6.10 References 146 Chapter Further engineering of AtdA 149 7.1 Introduction 149 7.2 Materials and methods 155 vi 7.2.1 Materials 155 7.2.2 Saturation mutagenesis 155 7.2.3 Random mutagenesis by error prone PCR 156 7.2.4 Screening method 156 7.2.5 Whole cell activity assay 157 7.2.6 Sample Preparation for SDS-PAGE Analysis 157 7.3 Second round of saturation mutagenesis 157 7.4 SDS-PAGE analysis of mutant 2-A21 160 7.5 Whole cell activity of mutant 2-A21 160 7.6 Third round of saturation mutagenesis 164 7.7 Directed evolution of AtdA3 by random mutagenesis 164 7.7.1 Mutation Rate of epPCR library 164 7.7.2 Random mutagenesis of AtdA3 165 7.8 SDS-PAGE analysis of mutant 3-R21 166 7.9 Whole cell activity of mutant 3-R21 168 7.10 Structural analysis of mutation 171 7.11 Summary 173 7.12 References 176 Chapter Summary, conclusion and future work 179 8.1 Summary 179 8.2 Conclusion 183 8.3 Future work 183 8.4 References 186 Appendix A Sequences of Plasmid Constructs 187 A.1 pTA2-3 sequence 187 A.2 pACYC A1A2 sequence 190 A.3 pET A3A4A5 sequence 192 vii A.4 pACYC A1 sequence 195 A.5 pACYC A2 sequence 197 A.6 pET A4A5 sequence 199 viii Conclusion and future work 8.3 References Aharoni, A., A. D. Griffiths and D. S. Tawfik (2005). "High-throughput screens and selections of enzyme-encoding genes." Curr Opin Chem Biol (2): 210-6. Fukumori, F. and C. P. Saint (1997). "Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1)." J Bacteriol 179 (2): 399-408. Liang, Q., M. Takeo, M. Chen, W. Zhang, Y. Xu and M. Lin (2005). "Chromosomeencoded gene cluster for the metabolic pathway that converts aniline to TCA-cycle intermediates in Delftia tsuruhatensis AD9." Microbiology 151 (Pt 10): 3435-46. Murakami, S., T. Hayashi, T. Maeda, S. Takenaka and K. Aoki (2003). "Cloning and functional analysis of aniline dioxygenase gene cluster, from Frateuria species ANA-18, that metabolizes aniline via an ortho-cleavage pathway of catechol." Biosci Biotechnol Biochem 67 (11): 2351-8. Parales, J. V., R. E. Parales, S. M. Resnick and D. T. Gibson (1998). "Enzyme specificity of 2-nitrotoluene 2,3-dioxygenase from Pseudomonas sp. strain JS42 is determined by the C-terminal region of the alpha subunit of the oxygenase component." J Bacteriol 180 (5): 1194-9. Parales, R. E., M. D. Emig, N. A. Lynch and D. T. Gibson (1998). "Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems." J Bacteriol 180 (9): 2337-44. Takeo, M., T. Fujii and Y. Maeda (1998a). "Sequence analysis of the genes encoding a multicomponent dioxygenase involved in oxidation of aniline and o-toluidine in Acinetobacter sp. strain YAA." J Ferment Bioeng 85 (1): 17-24. Tan, H. M. and C. M. Cheong (1994). "Substitution of the ISP alpha subunit of biphenyl dioxygenase from Pseudomonas results in a modification of the enzyme activity." Biochem Biophys Res Commun 204 (2): 912-7. Tesmer, J. J., T. J. Klem, M. L. Deras, V. J. Davisson and J. L. Smith (1996). "The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families." Nat Struct Biol (1): 74-86. Urata, M., E. Uchida, H. Nojiri, T. Omori, R. Obo, N. Miyaura and N. Ouchiyama (2004). "Genes involved in aniline degradation by Delftia acidovorans strain 7N and its distribution in the natural environment." Biosci Biotechnol Biochem 68 (12): 2457-65. Yamashita, M. M., R. J. Almassy, C. A. Janson, D. Cascio and D. Eisenberg (1989). "Refined atomic model of glutamine synthetase at 3.5 A resolution." J Biol Chem 264 (30): 17681-90. 186 Appendix A Apendix A Sequences of Plasmid Constructs A.1 pTA2-3 sequence FEATURES atdA1 Location/Qualifiers 327 1826 atdA2 1845 2570 atdA3 2591 3868 atdA4 3889 4482 atdA5 4496 5503 Amp resistance 6050 6908 PBR322 origin 6975 7671 lacI 8259 9338 trc promoter 193 222 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 ttatcatcga gtatggctgt tctggataat tgttgacaat acacaggaaa gacaaaggaa agataagcag gatgattcgg agcagccctc caacctagtc tgagtttgat caaggtctta tggtgaacca cgatgaaggt tgatcgctca agttcaaccc cgatattatg agaagatgag agcagccgat ttatcacgca gcatatgcat aggggaagtg tgccgcctcg gcttgcacca ttctgcaaca cccttattta ggatcccggc tttggctgag cgacaccttt tgctgaaggt atacttcaac gtgctctgaa aaactccgac gtttgacagc ggaagctgtg gcactcccgt tgaaatgagc taacaatttc gagtcgacta atctttggac tagggcttga cgctgtcggt ctttctcttt tcggaattga ctacgacgtt attggaaatc aatcgttaag cgaaaattgt atgccattca atcaggtgga tgcgctcaat aaggtttaga cacgacatgg cttcaggctg taccctcaga caaatggtag agccgcactc tccgtgtaat ccggcgccaa aaatcaaaag tgccaacggc gctgctttgg gatttctcga aacaaaaaga cattattgtg ttaaaactga ctgcacggtg gcaggtcgta gttttttgcg taatcatccg cagaccatgc tgtcccatga cgagatgcag ctctcctggg aaggcggcat agcgaatggg gagttgggtg ccttgggcag ttcccattat tacttattta ctttctccag gtggcgcaag tccaaggttc ttggcaccaa gcagcgctac acatttatgt caatcactag gtatccccgc agtttcacaa gaccgaagag ggcgatccgg tatatggcat gggttgcaag gctctggatg attaaatatt gctgaggctg ttactgtgat gaagagcgct tgggaagtta caccaatgct aatcactgca ccgacatcat gctcgtataa cattcgagct gtgagaaatt ccgacaaagt ctgatcagta tctcagaagg ttttcaaccc gtgtcccgag ataaaaccgg gtccccgcgg aatgcggtat agagtttagg ggtactccta gaaaaggtct gccaaatgga ttataaaatc gcaagccggc tggataaaga taggtcgagc caccaactgt cttgggcgaa ctagccggat cacaaattgt gggctcctta ctcttgagca ggctgcaatt ctgagcctac tttcagaggt ttgattatcg taagtgcatt tctggcgtca taattcgtgt aacggttctg tgtgtggaat cggtacccgg agattttata tctcgcagaa tggtctcttg gtcagaagtt atttactgct tgtggtgatg ctggatgctg tatcatgaag tgagcttgaa tgcgccaggt tctacttgaa tctcgagctc aaccacgttt ggccatcaaa aattaacggg tacccgaaag ttatgctggt gaatgggtat agaaaacaag cgaaaatcgt ctctgggctt tggtgcacaa cgattcggag aagaagatcc aggtgctgtc caatatgtct agaagaaatg cacagactta ggcagccatc cgctcaaggc gcaaatattc tgtgagcgga ggatcctcta acgaaaaata attgattctt cgaggtaagg acgatggcac ggtggtggct gttccagatc gcagatctgc aaggctgtca tggtacttga gtacagcctg tatcacttag aatctgcctt gatgtaatgg caaatatgtt ttctctgttg aatcttttta ggattacttg cgaagacgtc gcagcgatgg attggtgagc gatggcatta gtaccaatgc ttgtttagaa gagtgggcaa acgcagtggg aaacgctttg gtaaatgcct aataaaatta 187 Appendix A 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 tcgataatta aaaagttttc ttggcatatg accctagtcg aacatgttgg ttagacgccc ttgcggtggg tggagcaaga gcaggtttca aagcgactct ggaacagacc ctatacagaa gtttctctta tgggcgacca ttgtccgcat ttgcccttac gaatgcctat ggaaagctat gcatcttggt taaagtatcg taatgctggt gcttcgatat cctttatgct gtcagggtgg taataatagt taatattttt tgttaatgaa caatgcaatt ggccaacttt ttttagcagg accaacatct agtaaacaaa aaagatctta attaagcagt cacaagataa gagtggttaa gcgtcacctg attgtaaggc atccttgggg acaagcttta ggttatgtaa aacgactgtc tacattaaaa tctgaagccg aatccgaact caatgaggac gtgtgacaat cccgcagaac aatatccatt taactcctct cccggatcgg cgcgtttgag ccccttcatg aaccaaggag agctgaaaaa ctccgaagat gtgctgtgct agaaagcaat atctaaaccg atgcaagctt agaacgcaga acctgacccc tccccatgcg cgatggcttt tggcttattt cttcggttgt tgagtttagg taccagtgaa acttggatct gccgtatgca ctttctacgg tgctgagctg acacaagcaa atgaaaacca gcatctattt catgcaagcc ctgatcgttg cgtggggcaa catggttgga ggagagggtt cgaggttttg agtgcgcgcc aagtctgttc gatggctacc ggcggcgggg ttgggtaatg gatcagcagc agccagccag ccaaacttac acagttctgc cggatgcgca gaatcatgtc catatgaatg gagatccata gagaatcagt gtataaaaat actctgatta atatgtttct cactgtttct ccagcgaggc tgcaaacggg cgccagaagt ttgtgtttga ttattaagga tttcgccgca tttcgagtta ttggacggtg gaaaaaggtc tttaaaatca gtaaaggttg tgggatcgag ataaaaacag gaaagttcta cttgatatac caatatattg ggaggtgtag tcttttgctg gatgtcactg gattttattt ggtaattgtg actgttttgg agatcgaaaa ggctgttttg agcggtctga atgccgaact agagtaggga gttatcagtg gaatttattc cagtcccttg tttggaactg gagcgagtga acattactcg gtcggtatcc gttcatctcg agtggttatc attaattttc taaatcaact ttcaagcgga aaattcctaa taagaaaagg aggtttgtcg aattcaggaa tcgataaaga tctttgctac agtatattga aacgttatga atgtcccttt atatacagta gtcactcagt gaccacagcc caagagattt tgttaattgg attggcatgc ctcaggagga aagaaggact aaggagaaaa gtcgccatta ctgaggttta tgttgatcct tttttggaat gactaaagaa ggaggatggc cacatatgag ttttgcctat atgggcagtg aagccgagat taatgatgag aggcaataat ttgataagat tcttggctga ttttatttcg cggtaaagag gcgactttat attttgtttt cgctaaatag taatatttca aattctggtt atgatgctct agaatttttt ggagtgtttc taaactttat taaacgagat ggtcttgcat acgcttctga atatagaaat gcggatgaga taaaacagaa cagaagtgaa actgccaggc gtagtgagta gagcggtcca ctgtcgcact atgagctcac ggcttattga cacgttctga aaggacatcc aagatggtaa agcctcctca agaacttggt aattcagtcc aatggacaaa acttgatgat agatgatgag aaatgattcg ctcaggcaaa caatttctcc cagtaatgag tgaatggtta aataaaatgt ttcccatcag tttcggtaat gatagatcag tggccgcgaa agagcgagcg caaccaaata aaccttgctt ttttccaatt cgagaccatg tgcttgctac ttttgatacc aaccggttga agcgctgtta ctttctaacc gctcggcttt tgttactgga tttcatgata tcgcagatac ccggggtcaa ggcaagtctc ttgaagataa tcattttttc agcggaaacg gcactcccct gtcttattcc agaaagggga cgagacactc ttttgcagga acacaaaaat taaagagtta agacgatgaa agaggttgaa gatcgagtct tgatgataat gctaaatggc aataaaagct gtgtctgctg tgaggaagac atccttcgac gaagattttc tttgcctggc acgccgtagc atcaaataaa ctcagttaat taagaaagaa tggcggagag gtttcaaaat aagccatgga ttcaactgct agagatcagt tttgcaagaa agcgatacgt gggtgatgta gggcgcgtac atatttcaag tatcaaacgg tttcaggcat ggtaactcca gcttttgtta atgacggcaa aacgctgttt gcccaccagg aattggaagc tcattgctac gccgatgaga cgcccggaaa agctatgaaa gttggtgctg caggttattg gctggtgata atgggtgagg ccggaaatcg caagatgtta tggctacagc ggtacgccat acctcgctta ttggtgagcc tggatcagca ttcctggcag tacgcagact ctgtttcaaa gcgaggggtt aagttttaag agatgaaaca tatagttggt aaggagtcgt ggcaagtatt ctatcttcgt ggcagagggt ccccctgctg ggtagtggta aggattaagt aaagatctat aaaggtattc tattttttat aaagtcccac ggcgatacag attaagaaca ggaattaatg gtgagtggag ggctggatct caataagtcg agcctgatac ggcagtagcg gccgatggta acgaaaggct gctgataaag aaaccaattg gtgggtttga ggacttaaca gaatgcgtca gtagaaattt aaaaaaaccc gatgaggtac caattagtga taagaattca accgtaaggt cgaactgggt ttcggatggg tgcttaatcg agacatttac ttcctggtgc ttccacgggt cgttagagga gtggtgagat tggtgtttga aaatgactac caggtatggg tgcataaaga cacacgttcg gaatgaatct atcctatttc atgaagagct tggatgacgt aatggatcga tacaacataa taatgtctgc gaataataat ttatcaagag tgcgcttgat gtgttttgat tatgccggcg gaaagatcga aactaatcgc tttggttaga tggttggtat gataaattta ctaaaatgaa tttcatttgt taccaattaa cagcttcggc cgaactggtt gcagtttcca taactcctgt tgttttatgc gtttacaatt caacctctat gtggacctgc ctggactgat ttgaaagctc gcgttatgtg ttcctagttc atgtaattct tggcgtgtcg acctgcaggc agattaaatc cggtggtccc gtgtggggtc cagtcgaaag 188 Appendix A 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 9061 9121 9181 9241 9301 9361 actgggcctt cgccgggagc cgccataaac cgtttctaca gagacaataa acatttccgt cccagaaacg catcgaactg tccaatgatg cgggcaagag accagtcaca cataaccatg ggagctaacc accggagctg ggcaacaacg attaatagac ggctggctgg tgcagcactg tcaggcaact gcattggtaa tttttaattt ttaacgtgag ttgagatcct agcggtggtt cagcagagcg caagaactct tgccagtggc ggcgcagcgg ctacaccgaa gagaaaggcg gcttccaggg tgagcgtcga cgcggccttt gttatcccct ccgcagccga gcggtatttt tacaatctgc tgggtcatgg ctgctcccgg aggttttcac aagcggcatg gatagcgccc atgtcgcaga gccacgtttc ttcccaaccg cctccagtct atcaactggg aagcggcggt tggatgacca ttgatgtctc gactgggcgt cattaagttc atcaaattca aaaccatgca agatggcgct tctcggtagt ccatcaaaca ctcagggcca ccaccctggc agctggcacg agttagcgcg tcgttttatc ggatttgaac tgccaggcat aactcttttt ccctgataaa gtcgccctta ctggtgaaag gatctcaaca agcactttta caactcggtc gaaaagcatc agtgataaca gcttttttgc aatgaagcca ttgcgcaaac tggatggagg tttattgctg gggccagatg atggatgaac ctgtcagacc aaaaggatct ttttcgttcc ttttttctgc tgtttgccgg cagataccaa gtagcaccgc gataagtcgt tcgggctgaa ctgagatacc gacaggtatc ggaaacgcct tttttgtgat ttacggttcc gattctgtgg acgaccgagc ctccttacgc tctgatgccg ctgcgccccg catccgctta cgtcatcacc catttacgtt ggaagagagt gtatgccggt tgcgaaaacg cgtggcacaa ggccctgcac tgccagcgtg gcacaatctt ggatgccatt tgaccagaca ggagcatctg tgtctcggcg gccgatagcg aatgctgaat gggcgcaatg gggatacgac ggattttcgc ggcggtgaag gcccaatacg acaggtttcc aattgatctg tgttgtttgt gttgcgaagc caaattaagc gtttattttt tgcttcaata ttcccttttt taaaagatgc gcggtaagat aagttctgct gccgcataca ttacggatgg ctgcggccaa acaacatggg taccaaacga tattaactgg cggataaagt ataaatctgg gtaagccctc gaaatagaca aagtttactc aggtgaagat actgagcgtc gcgtaatctg atcaagagct atactgtcct ctacatacct gtcttaccgg cggggggttc tacagcgtga cggtaagcgg ggtatcttta gctcgtcagg tggccttttg ataaccgtat gcagcgagtc atctgtgcgg catagttaag acacccgcca cagacaagct gaaacgcgcg gacaccatcg caattcaggg gtctcttatc cgggaaaaag caactggcgg gcgccgtcgc gtggtgtcga ctcgcgcaac gctgtggaag cccatcaaca gtcgcattgg cgtctgcgtc gaacgggaag gagggcatcg cgcgccatta gataccgaag ctgctggggc ggcaatcagc caaaccgcct cgactggaaa cggtgaacgc aacggcccgg agaaggccat ctaaatacat atattgaaaa tgcggcattt tgaagatcag ccttgagagt atgtggcgcg ctattctcag catgacagta cttacttctg ggatcatgta cgagcgtgac cgaactactt tgcaggacca agccggtgag ccgtatcgta gatcgctgag atatatactt cctttttgat agaccccgta ctgcttgcaa accaactctt tctagtgtag cgctctgcta gttggactca gtgcacacag gctatgagaa cagggtcgga tagtcctgtc ggggcggagc ctggcctttt taccgccttt agtgagcgag tatttcacac ccagtataca acacccgctg gtgaccgtct aggcagcaga aatggtgcaa tggtgaatgt agaccgtttc tggaagcggc gcaaacagtc aaattgtcgc tggtagaacg gcgtcagtgg ctgcctgcac gtattatttt gtcaccagca tggctggctg gcgactggag ttcccactgc ccgagtccgg acagctcatg aaaccagcgt tgttgcccgt ctccccgcgc gcgggcagtg tctcctgagt agggtggcgg cctgacggat tcaaatatgt aggaagagta tgccttcctg ttgggtgcac tttcgccccg gtattatccc aatgacttgg agagaattat acaacgatcg actcgccttg accacgatgc actctagctt cttctgcgct cgtgggtctc gttatctaca ataggtgcct tagattgatt aatctcatga gaaaagatca acaaaaaaac tttccgaagg ccgtagttag atcctgttac agacgatagt cccagcttgg agcgccacgc acaggagagc gggtttcgcc ctatggaaaa gctcacatgt gagtgagctg gaagcggaag cgcatatggt ctccgctatc acgcgccctg ccgggagctg tcaattcgcg aacctttcgc gaaaccagta ccgcgtggtg gatggcggag gttgctgatt ggcgattaaa aagcggcgtc gctgatcatt taatgttccg ctcccatgaa aatcgcgctg gcataaatat tgccatgtcc gatgctggtt gctgcgcgtt ttatatcccg ggaccgcttg ctcactggtg gttggccgat agcgcaacgc aggacaaatc gcaggacgcc ggcctttttg atccgctcat tgagtattca tttttgctca gagtgggtta aagaacgttt gtgttgacgc ttgagtactc gcagtgctgc gaggaccgaa atcgttggga ctacagcaat cccggcaaca cggcccttcc gcggtatcat cgacggggag cactgattaa taaaacttca ccaaaatccc aaggatcttc caccgctacc taactggctt gccaccactt cagtggctgc taccggataa agcgaacgac ttcccgaagg gcacgaggga acctctgact acgccagcaa tctttcctgc ataccgctcg agcgcctgat gcactctcag gctacgtgac acgggcttgt catgtgtcag cgcgaaggcg ggtatggcat acgttatacg aaccaggcca ctgaattaca ggcgttgcca tctcgcgccg gaagcctgta aactatccgc gcgttatttc gacggtacgc ttagcgggcc ctcactcgca ggttttcaac gccaacgatc ggtgcggata ccgttaacca ctgcaactct aaaagaaaaa tcattaatgc aattaatgtg // 189 Appendix A A.2 pACYC A1A2 sequence FEATURES lacI Location/Qualifiers complement(4903 5982) Cm resistance complement(2846 3502) P15A origin 3864 4776 T7 promoter 6106 6122 T7 promoter 1699 1715 atdA1 71 1618 atdA2 1785 2543 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 gagcggataa atgggcagca ttagatttta gttctcgcag tatggtctct gggtcagaag ccatttactg agtgtggtga ggctggatgc ggtatcatga attgagcttg ggtgcgccag tatctacttg cttctcgagc gaaaccacgt tcggccatca gcaattaacg gatacccgaa gcttatgctg gtgaatgggt aaagaaaaca atcgaaaatc gtctctgggc tatggtgcac cacgattcgg ttaagaagat acaggtgctg cttgcggccg atcgaaatta gtatattagt cggccacatg tcgagaagaa attcacagac gtactcagtt ccataagaaa acttggcgga cacgtttcaa tgaaagccat tgattcaact tccagagatc taatttgcaa tcaagcgata ggtgggtgat ggggcctcta acggtcacac ggggaattgt gagatatacc gagtgagaaa agccgacaaa ggctgatcag attctcagaa ggttttcaac tggtgtcccg agataaaacc atgtccccgc taaatgcggt agagagttta agggtactcc tcgaaaaggt aagccaaatg acttataaaa gtgcaagccg agtggataaa gctaggtcga aacaccaact agcttgggcg ggctagccgg atcacaaatt aggggctcct tgctcttgag ttggctgcaa tgctgagcct agtcgacaag cggccgcata ccccatctta ggatatcggc gctttgatta ttataagtgc gtggtagtga ttcgagcggt ttgctgtcgc ctgatgagct tgaggcttat tcgcacgttc tccaaggaca tcgaagatgg atcagcctcc ttcagaactt ataacccctt tgagaagcac caattcccct gccatcacca taacgaaaaa aaattgattc tgcgaggtaa ttacgatggc ctggtggtgg tggttccaga tggcagatct agaaggctgt aatggtactt gtgtacagcc aatatcactt tcaatctgcc ttgatgtaat aacaaatatg ggttctctgt agaatctttt gtggattact atcgaagacg aggcagcgat gtattggtga ttgatggcat aagtaccaat agttgtttag ccgagtgggc tcacgcagtg cataatgctt atacgactca taagtataag tctaaacgct atggtaaatg ttaaataaaa aatgctgata gaaaaaccaa gaggtgggtt aatggactta ggagaatgcg gctgtagaaa agtaaaaaaa gaagatgagg cgtcaattag gtataaccta aacgggtctt tgcttccggt gtagaaataa tcatcaccac taatctttgg tttagggctt ggcgctgtcg acctttctct cttcggaatt tcctacgacg gcattggaaa caaatcgtta gacgaaaatt tgatgccatt agatcaggtg tttgcgctca ggaaggttta ttcacgacat tgcttcaggc tataccctca tgcaaatggt tcagccgcac ggtccgtgta gcccggcgcc taaaatcaaa gctgccaacg aagctgcttt aagatttctc ggaacaaaaa aagtcgaaca ctatagggga aaggagatat ttgcattatt cctttaaaac ttatcgataa aagaaaagtt ttgttggcat tgaaccctag acaaacatgt tcattagacg tttttgcggt ccctggagca tacgcaggtt tgaaagcgac ggctgctgcc gaggggtttt agtcaataaa ttttgtttaa agccaggatc acagataagc gagatgattc gtagcagccc ttcaacctag gatgagtttg ttcaaggtct tctggtgaac agcgatgaag gttgatcgct caagttcaac gacgatatta atagaagatg gaagcagccg ggttatcacg tggcatatgc gaaggggaag agtgccgcct tcgcttgcac atttctgcaa aacccttatt agggatcccg gctttggctg ggcgacacct gatgctgaag gaatacttca gaaagtaatc attgtgagcg acatatggca gtggtgctct tgaaaactcc ttacgatggc ttctggctta atgcttcggt tcgtgagttt tggtaccagt cccacttgga ggggccgtat agactttcta tcatgctgag tctacacaag accgctgagc ttgctgaaac ccggtaaacc ctttaataag cgaattcgat agcgagatgc ggctctcctg tcaaggcggc tcagcgaatg atgagttggg taccttgggc cattcccatt gttacttatt cactttctcc ccgtggcgca tgtccaaggt agttggcacc atgcagcgct caacatttat atcaatcact tggtatcccc cgagtttcac cagaccgaag caggcgatcc tatatatggc gcgggttgca aggctctgga ttattaaata gtgctgaggc acttactgtg gtattgtaca gataacaatt gatctcaatt gaagaagagc gactgggaag tttgttatca tttgaattta tgtcagtccc aggtttggaa gaagagcgag tctacattac gcagtcggta cgggttcatc ctgagtggtt caaattaatt aataactagc ctcaggcatt agcaatagac 190 Appendix A 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 ataagcggct tttctgccat accaataact attcattaag ccagcggcat cgaagaagtt tggctgagac cgtaacacgc cactccagag cactatccca tcatcaggcg cggtctttaa ctgactgaaa atccagtgat aaaatacgcc gatcaacgtc caggatttat cgtcgggtga gtggcttctg ggcaaaagca ctgatgaggg tcagcagaat ggtcgttcga tgccaggaag ctccgccccc acaggactat ctgcctttcg ctgacactca gttcagtccg catgcaaaag gtcatgcgcc ccagttacct ggcggttttt tcatcttatt agcacctgaa gcccaccgga gcctaatgag ggaaacctgt cgtattgggc cttcaccgcc gcgaaaatcc gtcgtatccc cattgcgccc attcagcatt cgctatcggc cgccgagaca cagatgctcc tgtctggtca aatggcatcc aagattgtgc cacgctggca gtgcagggcc ttgtgccacg cgttttcgca accggcatac actctcttcc cgggatctcg ta atttaacgac tcatccgctt gccttaaaaa cattctgccg cagcaccttg gtccatattg gaaaaacata cacatcttgc cgatgaaaac tatcaccagc ggcaagaatg aaaggccgta tgcctcaaaa ttttttctcc cggtagtgat tcattttcgc ttattctgcg tgctgccaac tttctatcag ccgccggaca tgtcagtgaa atgtgataca ctgcggcgag atacttaaca ctgacaagca aaagatacca gtttaccggt gttccgggta accgctgcgc caccactggc ggttaaggct cggttcaaag tcgttttcag aatcagataa gtcagcccca aggagctgac tgagctaact cgtgccagct gccagggtgg tggccctgag tgtttgatgg actaccgaga agcgccatct tgcatggttt tgaatttgat gaacttaatg acgcccagtc gagacatcaa tggtcatcca accgccgctt cccagttgat agactggagg cggttgggaa gaaacgtggc tctgcgacat gggcgctatc acgctctccc cctgccctga attatcactt aattacgccc acatggaagc tcgccttgcg gccacgttta ttctcaataa gaatatatgt gtttcagttt tcaccgtctt tgaataaagg atatccagct tgttctttac attttagctt cttatttcat caaaagttgg aagtgatctt ttactgattt ctgtccctcc tcagcgctag gtgcttcatg ggatatattc cggaaatggc gggaagtgag tcacgaaatc ggcgtttccc gtcattccgc ggcagttcgc cttatccggt agcagccact aaactgaaag agttggtagc agcaagagat aatatttcta tacgatataa tgggttgaag tacattaatt gcattaatga tttttctttt agagttgcag tggttaacgg tgtccgcacc gatcgttggc gttgaaaacc tgcgagtgag ggcccgctaa gcgtaccgtc gaaataacgc gcggatagtt tacaggcttc cggcgcgaga tggcaacgcc tgtaattcag tggcctggtt cgtataacgt atgccatacc ttatgcgact accgacgacc attcaggcgt cgccctgcca catcacagac tataatattt aatcaaaact accctttagg gtagaaactg gctcatggaa tcattgccat ccggataaaa gaacggtctg gatgccattg ccttagctcc tatggtgaaa cccagggctt ccgtcacagg agtgtatgat tgttcagcta cggagtgtat tggcaggaga cgcttcctcg ttacgaacgg agggccgcgg tgacgctcaa ctggcggctc tgttatggcc tccaagctgg aactatcgtc ggtaattgat gacaagtttt tcagagaacc tacgcgcaga gatttcagtg gttgtaattc gctctcaagg gcgttgcgct atcggccaac caccagtgag caagcggtcc cgggatataa aacgcgcagc aaccagcatc ggacatggca atatttatgc cagcgcgatt ttcatgggag cggaacatta aatgatcagc gacgccgctt tttaatcgcc aatcagcaac ctccgccatc caccacgcgg tactggtttc gcgaaaggtt cctgcattag gggtcgaatt agcaccaggc ctcatcgcag ggcatgatga gcccatagtg ggtgaaactc gaaataggcc ccggaaatcg aacggtgtaa acggaactcc cttgtgctta gttataggta ggatatatca tgaaaatctc gttggaacct cccggtatca tatttattcg ggtgtttttg ctgacggggt actggcttac aaaaaggctg ctcactgact ggcggagatt caaagccgtt atcagtggtg cctcgtgcgc gcgtttgtct actgtatgca ttgagtccaa ttagaggagt ggtgactgcg ttcgaaaaac ccaaaacgat caatttatct tcatgttagt gcatcggtcg cactgcccgc gcgcggggag acgggcaaca acgctggttt catgagctgt ccggactcgg gcagtgggaa ctccagtcgc cagccagcca tgctggtgac aaaataatac gtgcaggcag ccactgacgc cgttctacca gcgacaattt gactgtttgc gccgcttcca gaaacggtct acattcacca ttgcgccatt gaaattaata tgctttcgaa gtttaagggc tactgttgta acctgaatcg aaaacggggg acccagggat aggttttcac tcgtggtatt caagggtgaa ggatgagcat tttttcttta cattgagcaa acggtggtat gataactcaa cttacgtgcc acagggacac gcgcaaagtg aggtgctcca ggtgcgtaac tatgttggca caccggtgcg cgctacgctc tcctggaaga tttccatagg gcgaaacccg tctcctgttc cattccacgc cgaacccccc cccggaaaga tagtcttgaa ctcctccaag cgccctgcaa ctcaagaaga cttcaaatgt catgccccgc agatcccggt tttccagtcg aggcggtttg gctgattgcc gccccagcag cttcggtatc taatggcgcg cgatgccctc cttcccgttc gacgcagacg ccaatgcgac tgttgatggg cttccacagc gttgcgcgag tcgacaccac gcgacggcgc ccgccagttg ctttttcccg gataagagac ccctgaattg cgatggtgtc cgactcacta // 191 Appendix A A.3 pET A3A4A5 sequence FEATURES lacI Location/Qualifiers complement(6712 7794) PBR322 origin 5518 5519 Amp resistance 3900 4757 T7 promoter 8185 8201 T7 promoter 1477 1493 atdA3 71 1396 atdA4 1563 2189 atdA5 2203 3210 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 gagcggataa atgggcagca aatcaactaa caagcggaaa attcctaaac agaaaaggag gtttgtcgaa ttcaggaact gataaagaca tttgctacca tatattgatg cgttatgaaa gtcccttttt atacagtatt cactcagtga ccacagcctg agagatttag ttaattggca tggcatgcaa caggaggatt gaaggactcg ggagaaaatg cgccattatt gaggtttaag attgtacacg taacaattcc tctcaattgg tgatcctagc ttggaatctt taaagaagct ggatggctgt atatgagttt tgcctattcg ggcagtgccg ccgagatggc tgatgagttg caataattca ataagatagc tggctgagca tatttcggtc taaagagaga actttatcga ttgttttttt ggggaattgt gagatatacc gaaaaccata atctattttt tgcaagccaa gatcgttgta tggggcaaag tggttggaaa agagggtttc aggttttgtc tgcgcgccag gtctgttcaa tggctaccat cggcggggat gggtaatggt tcagcagcga ccagccagca aaacttactg agttctgcat gatgcgcact atcatgtcaa tatgaatgaa gatccatagt gaatcagtct aagtaatcgt tgtgagcgga atatggcaga taaaaattgt ctgattattt tgtttctgac tgtttctgga gcgaggccac aaacgggttt cagaagtatg tgtttgaaag ttaaggataa cgccgcaagg cgagttattg gacggtgtct aaaggtcttt aaaatcacgg aaggttggcg gatcgagatt caattcccct gccatcacca ttcagtccgg tggacaaaat ttgatgatta atgatgagtt atgattcggg caggcaaagc atttctccat gtaatgagaa aatggttagc taaaatgtaa cccatcagtc tcggtaatgc tagatcagcg gccgcgaaag agcgagcggt accaaataca ccttgcttgc ttccaattat agaccatgcc cttgctacca ttgatacctg tcgacaagct gccgcataat ccatcttagt atatcggccg gctgttaacc tctaaccttg cggcttttgg tactggattc catgatatac cagatacctg gggtcaagcg aagtctcaag aagataaaga ttttttctat ggaaacgaag ctcccctggc ttattcccta aaggggaggc gacactcccc tgcaggaggt ctagaaataa tcatcaccac gcgcgtacac atttcaagcg tcaaacggtt tcaggcattg taactccaag ttttgttatt gacggcaatt cgctgtttcg ccaccagggt ttggaagctg attgctacaa cgatgagaca cccggaaatg ctatgaaaca tggtgctgga ggttattgat tggtgataat gggtgaggtg ggaaatcgaa agatgttata gctacagcta tgcggccgca cgaaattaat atattagtta gccacatgaa tcgcttatta gtgagcctgc atcagcagtg ctggcagtat gcagactgaa tttcaaaaac aggggttttt ttttaagtgg tgaaacagat agttggtcta gagtcgtttt aagtatttac tcttcgtcag agagggtcga cctgctggca agtggtataa ttttgtttaa agccaggatc cgtaaggtct aactgggtgt cggatgggtg cttaatcgtt acatttactt cctggtgcga ccacgggtgg ttagaggagc ggtgagatta gtgtttgata atgactacgc ggtatgggcc cataaagagt cacgttcgta atgaatctta cctatttctg gaagagctca gatgacgtgg tggatcgatt caacataaac atgtctgcag taatgcttaa acgactcact agtataagaa taataataaa tcaagagatt gcttgatcac ttttgatgag gccggcggcg agatcgaatt taatcgcatc ggttagaaca ttggtatggt aaatttaaac aaatgaatac catttgttct caattaaaat cttcggccaa actggttgtg gtttccaccc ctcctgtaat ctttaagaag cgaattcgat atacagaagc ttctcttaca ggcgaccact gtccgcatcg gcccttacca atgcctatgg aaagctatcg atcttggtag aagtatcgaa atgctggtga ttcgatatgg tttatgcttt cagggtggga ataatagtag atatttttcc ttaatgaaac atgcaattcg ccaactttga ttagcaggca caacatctga taaacaaaga gtcgaacaga ataggggaat ggagatatac gatcttagta aagcagtact aagataaata tggttaacac tcacctgcca gtaaggctgc cttggggcgc agctttattg tatgtaatta gactgtcttt attaaaattt gaagccgttg ccgaactgaa tgaggacttt tgacaatgta gcagaactgg atccattata 192 Appendix A 2581 2641 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 aaaacagcgc agttctataa gatatacaat tatattgatg ggtgtagaga tttgctggga gtcactgtaa tttattttaa aattgtgggt gttttggacg tcgaaaaata aactagcata gaactatatc tgtggtggtt cgctttcttc ggggctccct ttagggtgat gttggagtcc tatctcggtc aaatgagctg ttctggcggc taaaaatgaa caatgcttaa gcctgactcc gctgcaatga ccagccggaa attaattgtt gttgccattg tccggttccc agctccttcg gttatggcag actggtgagt tgcccggcgt attggaaaac tcgatgtaac tctgggtgag aaatgttgaa ttgtctcatg ccaaaatccc aaggatcttc caccgctacc taactggctt gccaccactt cagtggctgc taccggataa agcgaacgac ttcccgaagg gcacgaggga acctctgact acgccagcaa tctttcctgc ataccgctcg agcgcctgat gtgcactctc tcgctacgtg tgacgggctt tgcatgtgtc tcatcagcgt ttgagtttct gttttttcct atgataccga cggttactgg aaaatcactc taaatagaca tatttcataa tctggttaga atgctctaga attttttgat gtgtttctga actttatgct acgagataat cttgcatgtg cttctgatga tagaaatatc accccttggg cggattggcg acgcgcagcg ccttcctttc ttagggttcc ggttcacgta acgttcttta tattcttttg atttaacaaa acgatggcat gttttaaatc tcagtgaggc ccgtcgtgta taccgcgaga gggccgagcg gccgggaagc ctacaggcat aacgatcaag gtcctccgat cactgcataa actcaaccaa caatacggga gttcttcggg ccactcgtgc caaaaacagg tactcatact agcggataca ttaacgtgag ttgagatcct agcggtggtt cagcagagcg caagaactct tgccagtggc ggcgcagcgg ctacaccgaa gagaaaggcg gcttccaggg tgagcgtcga cgcggccttt gttatcccct ccgcagccga gcggtatttt agtacaatct actgggtcat gtctgctccc agaggttttc ggtcgtgaag ccagaagcgt gtttggtcac tgaaacgaga aacgttgtga agggtcaatg caaaaatagg agagttaaaa cgatgaaaaa ggttgaatat cgagtctaaa tgataatggc aaatggcatt aaaagctgga tctgctggtg ggaagacggc cttcgaccaa gcctctaaac aatgggacgc tgaccgctac tcgccacgtt gatttagtgc gtgggccatc atagtggact atttataagg aatttaacgc gagattatca aatctaaagt acctatctca gataactacg cccacgctca cagaagtggt tagagtaagt cgtggtgtca gcgagttaca cgttgtcaga ttctcttact gtcattctga taataccgcg gcgaaaactc acccaactga aaggcaaaat cttccttttt tatttgaatg ttttcgttcc ttttttctgc tgtttgccgg cagataccaa gtagcaccgc gataagtcgt tcgggctgaa ctgagatacc gacaggtatc ggaaacgcct tttttgtgat ttacggttcc gattctgtgg acgaccgagc ctccttacgc gctctgatgc ggctgcgccc ggcatccgct accgtcatca cgattcacag taatgtctgg tgatgcctcc gaggatgctc gggtaaacaa ccagcgcttc attaagttgt gatctatgtt ggtattccaa tttttatgtg gtcccacctg gatacagttg aagaacagcg attaatgttc agtggagatg tggatcttgg taacctaggc gggtcttgag gccctgtagc acttgccagc cgccggcttt tttacggcac gccctgatag cttgttccaa gattttgccg gaattttaac aaaaggatct atatatgagt gcgatctgtc atacgggagg ccggctccag cctgcaactt agttcgccag cgctcgtcgt tgatccccca agtaagttgg gtcatgccat gaatagtgta ccacatagca tcaaggatct tcttcagcat gccgcaaaaa caatcatgat tatttagaaa actgagcgtc gcgtaatctg atcaagagct atactgtcct ctacatacct gtcttaccgg cggggggttc tacagcgtga cggtaagcgg ggtatcttta gctcgtcagg tggccttttg ataaccgtat gcagcgagtc atctgtgcgg cgcatagtta cgacacccgc tacagacaag ccgaaacgcg atgtctgcct cttctgataa gtgtaagggg acgatacggg ctggcggtat gttaatacag tttatgctaa tacaattccc cctctatcgc gacctgcccc gactgataac aaagctcagc ttatgtgctc ctagttcgtg taattctaga cgtgtcgatc tgctgccacc gggttttttg ggcgcattaa gccctagcgc ccccgtcaag ctcgacccca acggtttttc actggaacaa atttcggcct aaaatattaa tcacctagat aaacttggtc tatttcgttc gcttaccatc atttatcagc tatccgcctc ttaatagttt ttggtatggc tgttgtgcaa ccgcagtgtt ccgtaagatg tgcggcgacc gaactttaaa taccgctgtt cttttacttt agggaataag tgaagcattt aataaacaaa agaccccgta ctgcttgcaa accaactctt tctagtgtag cgctctgcta gttggactca gtgcacacag gctatgagaa cagggtcgga tagtcctgtc ggggcggagc ctggcctttt taccgccttt agtgagcgag tatttcacac agccagtata caacacccgc ctgtgaccgt cgaggcagct gttcatccgc agcgggccat gatttctgtt ttactgatga ggatgcggcg atgtaggtgt ctcctctgaa ggatcggctt gtttgagcaa cttcatggga caaggagtct tgaaaaagat cgaagatgat ctgtgctggt aagcaatact taaaccgaga gctgagcaat ctgaaaggag gcgcggcggg ccgctccttt ctctaaatcg aaaaacttga gccctttgac cactcaaccc attggttaaa cgtttacaat ccttttaaat tgacagttac atccatagtt tggccccagt aataaaccag catccagtct gcgcaacgtt ttcattcagc aaaagcggtt atcactcatg cttttctgtg gagttgctct agtgctcatc gagatccagt caccagcgtt ggcgacacgg atcagggtta taggtcatga gaaaagatca acaaaaaaac tttccgaagg ccgtagttag atcctgttac agacgatagt cccagcttgg agcgccacgc acaggagagc gggtttcgcc ctatggaaaa gctcacatgt gagtgagctg gaagcggaag cgcatatatg cactccgcta tgacgcgccc ctccgggagc gcggtaaagc gtccagctcg gttaagggcg catgggggta tgaacatgcc ggaccagaga tccacagggt 193 Appendix A 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 agccagcagc gtttccagac gacgttttgc ccagtaaggc gtcatgcccc cgagatcccg gctttccagt agaggcggtt cagctgattg ttgccccagc gtcttcggta ggtaatggcg aacgatgccc gccttcccgt cagacgcaga acccaatgcg actgttgatg agcttccaca gcgttgcgcg catcgacacc ttgcgacggc gcccgccagt cactttttcc ctgataagag caccctgaat ttcgatggtg ccagtagtag tggcgcccaa tcatgagccc cagcaaccgc agatcgatct atcctgcgat tttacgaaac agcagcagtc aaccccgcca gcgcccaccg gtgcctaatg cgggaaacct tgcgtattgg cccttcaccg aggcgaaaat tcgtcgtatc cgcattgcgc tcattcagca tccgctatcg cgcgccgaga accagatgct ggtgtctggt gcaatggcat agaagattgt accacgctgg gcgtgcaggg tgttgtgcca cgcgttttcg acaccggcat tgactctctt tccgggatct gttgaggccg cagtcccccg gaagtggcga acctgtggcg cgatcccgcg gcagatccgg acggaaaccg gcttcacgtt gcctagccgg gaaggagctg agtgagctaa gtcgtgccag gcgccagggt cctggccctg cctgtttgat ccactaccga ccagcgccat tttgcatggt gctgaatttg cagaacttaa ccacgcccag cagagacatc cctggtcatc gcaccgccgc cacccagttg ccagactgga cgcggttggg cagaaacgtg actctgcgac ccgggcgcta cgacgctctc ttgagcaccg gccacggggc gcccgatctt ccggtgatgc aaattaatac aacataatgg aagaccattc cgctcgcgta gtcctcaacg actgggttga cttacattaa ctgcattaat ggtttttctt agagagttgc ggtggttaac gatgtccgca ctgatcgttg ttgttgaaaa attgcgagtg tgggcccgct tcgcgtaccg aagaaataac cagcggatag tttacaggct atcggcgcga ggtggcaacg aatgtaattc gctggcctgg atcgtataac tcatgccata ccttatgcga ccgccgcaag ctgccaccat ccccatcggt cggccacgat gactcactat tgcagggcgc atgttgttgc tcggtgattc acaggagcac aggctctcaa ttgcgttgcg gaatcggcca ttcaccagtg agcaagcggt ggcgggatat ccaacgcgca gcaaccagca ccggacatgg agatatttat aacagcgcga tcttcatggg gccggaacat ttaatgatca tcgacgccgc gatttaatcg ccaatcagca agctccgcca ttcaccacgc gttactggtt ccgcgaaagg ctcctgcatt gaatggtgca acccacgccg gatgtcggcg gcgtccggcg a tgacttccgc tcaggtcgca attctgctaa gatcatgcta gggcatcggt ctcactgccc acgcgcgggg agacgggcaa ccacgctggt aacatgagct gcccggactc tcgcagtggg cactccagtc gccagccagc tttgctggtg agaaaataat tagtgcaggc gcccactgac ttcgttctac ccgcgacaat acgactgttt tcgccgcttc gggaaacggt tcacattcac ttttgcgcca aggaagcagc tgcaaggaga aaacaagcgc atataggcgc tagaggatcg // 194 Appendix A A.4 pACYC A1 sequence FEATURES lacI Location/Qualifiers complement(4274 5353) Cm resistance complement(2217 2873) P15A origin 3235 4147 T7 promoter 5477 5493 T7 promoter 1699 1715 atdA1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 ggggaattgt gagatatacc gagtgagaaa agccgacaaa ggctgatcag attctcagaa ggttttcaac tggtgtcccg agataaaacc atgtccccgc taaatgcggt agagagttta agggtactcc tcgaaaaggt aagccaaatg acttataaaa gtgcaagccg agtggataaa gctaggtcga aacaccaact agcttgggcg ggctagccgg atcacaaatt aggggctcct tgctcttgag ttggctgcaa tgctgagcct agtcgacaag cggccgcata ccccatctta ggatatcggc ctgctgcgaa aggctgctgc tgaggggttt tagtcaataa aaccgacgac tattcaggcg ccgccctgcc ccatcacaga gtataatatt aaatcaaaac aaccctttag tgtagaaact tgctcatgga ttcattgcca gccggataaa tgaacggtct 71 1618 gagcggataa atgggcagca ttagatttta gttctcgcag tatggtctct gggtcagaag ccatttactg agtgtggtga ggctggatgc ggtatcatga attgagcttg ggtgcgccag tatctacttg cttctcgagc gaaaccacgt tcggccatca gcaattaacg gatacccgaa gcttatgctg gtgaatgggt aaagaaaaca atcgaaaatc gtctctgggc tatggtgcac cacgattcgg ttaagaagat acaggtgctg cttgcggccg atcgaaatta gtatattagt cggccacgcg atttgaacgc caccgctgag tttgctgaaa accggtaaac cgggtcgaat tagcaccagg actcatcgca cggcatgatg tgcccatagt tggtgaaact ggaaataggc gccggaaatc aaacggtgta tacggaactc acttgtgctt ggttataggt caattcccct gccatcacca taacgaaaaa aaattgattc tgcgaggtaa ttacgatggc ctggtggtgg tggttccaga tggcagatct agaaggctgt aatggtactt gtgtacagcc aatatcactt tcaatctgcc ttgatgtaat aacaaatatg ggttctctgt agaatctttt gtggattact atcgaagacg aggcagcgat gtattggtga ttgatggcat aagtaccaat agttgtttag ccgagtgggc tcacgcagtg cataatgctt atacgactca taagtataag atcgctgacg cagcacatgg caataactag cctcaggcat cagcaataga ttgctttcga cgtttaaggg gtactgttgt aacctgaatc gaaaacgggg cacccaggga caggttttca gtcgtggtat acaagggtga cggatgagca atttttcttt acattgagca gtagaaataa tcatcaccac taatctttgg tttagggctt ggcgctgtcg acctttctct cttcggaatt tcctacgacg gcattggaaa caaatcgtta gacgaaaatt tgatgccatt agatcaggtg tttgcgctca ggaaggttta ttcacgacat tgcttcaggc tataccctca tgcaaatggt tcagccgcac ggtccgtgta gcccggcgcc taaaatcaaa gctgccaacg aagctgcttt aagatttctc ggaacaaaaa aagtcgaaca ctatagggga aaggagatat tcggtaccct actcgtctac cataacccct ttgagaagca cataagcggc atttctgcca caccaataac aattcattaa gccagcggca gcgaagaagt ttggctgaga ccgtaacacg tcactccaga acactatccc ttcatcaggc acggtcttta actgactgaa ttttgtttaa agccaggatc acagataagc gagatgattc gtagcagccc ttcaacctag gatgagtttg ttcaaggtct tctggtgaac agcgatgaag gttgatcgct caagttcaac gacgatatta atagaagatg gaagcagccg ggttatcacg tggcatatgc gaaggggaag agtgccgcct tcgcttgcac atttctgcaa aacccttatt agggatcccg gctttggctg ggcgacacct gatgctgaag gaatacttca gaaagtaatc attgtgagcg acatatggca cgagtctggt tagcgcagct tggggcctct cacggtcaca tatttaacga ttcatccgct tgccttaaaa gcattctgcc tcagcacctt tgtccatatt cgaaaaacat ccacatcttg gcgatgaaaa atatcaccag gggcaagaat aaaaggccgt atgcctcaaa ctttaataag cgaattcgat agcgagatgc ggctctcctg tcaaggcggc tcagcgaatg atgagttggg taccttgggc cattcccatt gttacttatt cactttctcc ccgtggcgca tgtccaaggt agttggcacc atgcagcgct caacatttat atcaatcact tggtatcccc cgagtttcac cagaccgaag caggcgatcc tatatatggc gcgggttgca aggctctgga ttattaaata gtgctgaggc acttactgtg gtattgtaca gataacaatt gatctcaatt aaagaaaccg taattaacct aaacgggtct ctgcttccgg ccctgccctg tattatcact aaattacgcc gacatggaag gtcgccttgc ggccacgttt attctcaata cgaatatatg cgtttcagtt ctcaccgtct gtgaataaag aatatccagc atgttcttta 195 Appendix A 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 cgatgccatt tccttagctc ttatggtgaa gcccagggct tccgtcacag tagtgtatga ctgttcagct gcggagtgta gtggcaggag ccgcttcctc cttacgaacg gagggccgcg ctgacgctca cctggcggct ctgttatggc ctccaagctg taactatcgt tggtaattga ggacaagttt ctcagagaac ttacgcgcag agatttcagt agttgtaatt ggctctcaag tgcgttgcgc aatcggccaa tcaccagtga gcaagcggtc gcgggatata caacgcgcag caaccagcat cggacatggc gatatttatg acagcgcgat cttcatggga ccggaacatt taatgatcag cgacgccgct atttaatcgc caatcagcaa gctccgccat tcaccacgcg ttactggttt cgcgaaaggt tcctgcatta gggatatatc ctgaaaatct agttggaacc tcccggtatc gtatttattc tggtgttttt actgacgggg tactggctta aaaaaaggct gctcactgac gggcggagat gcaaagccgt aatcagtggt ccctcgtgcg cgcgtttgtc gactgtatgc cttgagtcca tttagaggag tggtgactgc cttcgaaaaa accaaaacga gcaatttatc ctcatgttag ggcatcggtc tcactgcccg cgcgcgggga gacgggcaac cacgctggtt acatgagctg cccggactcg cgcagtggga actccagtcg ccagccagcc ttgctggtga gaaaataata agtgcaggca cccactgacg tcgttctacc cgcgacaatt cgactgtttg cgccgcttcc ggaaacggtc cacattcacc tttgcgccat ggaaattaat aacggtggta cgataactca tcttacgtgc aacagggaca ggcgcaaagt gaggtgctcc tggtgcgtaa ctatgttggc gcaccggtgc tcgctacgct ttcctggaag ttttccatag ggcgaaaccc ctctcctgtt tcattccacg acgaaccccc acccggaaag ttagtcttga gctcctccaa ccgccctgca tctcaagaag tcttcaaatg tcatgccccg gagatcccgg ctttccagtc gaggcggttt agctgattgc tgccccagca tcttcggtat gtaatggcgc acgatgccct ccttcccgtt agacgcagac cccaatgcga ctgttgatgg gcttccacag cgttgcgcga atcgacacca tgcgacggcg cccgccagtt actttttccc tgataagaga accctgaatt tcgatggtgt acgactcact tatccagtga aaaaatacgc cgatcaacgt ccaggattta gcgtcgggtg agtggcttct cggcaaaagc actgatgagg gtcagcagaa cggtcgttcg atgccaggaa gctccgcccc gacaggacta cctgcctttc cctgacactc cgttcagtcc acatgcaaaa agtcatgcgc gccagttacc aggcggtttt atcatcttat tagcacctga cgcccaccgg tgcctaatga gggaaacctg gcgtattggg ccttcaccgc ggcgaaaatc cgtcgtatcc gcattgcgcc cattcagcat ccgctatcgg gcgccgagac ccagatgctc gtgtctggtc caatggcatc gaagattgtg ccacgctggc cgtgcagggc gttgtgccac gcgttttcgc caccggcata gactctcttc ccgggatctc ata tttttttctc ccggtagtga ctcattttcg tttattctgc atgctgccaa gtttctatca accgccggac gtgtcagtga tatgtgatac actgcggcga gatacttaac cctgacaagc taaagatacc ggtttaccgg agttccgggt gaccgctgcg gcaccactgg cggttaaggc tcggttcaaa ttcgttttca taatcagata agtcagcccc aaggagctga gtgagctaac tcgtgccagc cgccagggtg ctggccctga ctgtttgatg cactaccgag cagcgccatc ttgcatggtt ctgaatttga agaacttaat cacgcccagt agagacatca ctggtcatcc caccgccgct acccagttga cagactggag gcggttggga agaaacgtgg ctctgcgaca cgggcgctat gacgctctcc cattttagct tcttatttca ccaaaagttg gaagtgatct cttactgatt gctgtccctc atcagcgcta agtgcttcat aggatatatt gcggaaatgg agggaagtga atcacgaaat aggcgtttcc tgtcattccg aggcagttcg ccttatccgg cagcagccac taaactgaaa gagttggtag gagcaagaga aaatatttct atacgatata ctgggttgaa ttacattaat tgcattaatg gtttttcttt gagagttgca gtggttaacg atgtccgcac tgatcgttgg tgttgaaaac ttgcgagtga gggcccgcta cgcgtaccgt agaaataacg agcggatagt ttacaggctt tcggcgcgag gtggcaacgc atgtaattca ctggcctggt tcgtataacg catgccatac cttatgcgac // 196 Appendix A A.5 pACYC A2 sequence FEATURES lacI Location/Qualifiers complement(3418 4497) Cm resistance complement(1361 2017) P15A origin 2379 3291 T7 promoter 4621 4637 T7 promoter 214 230 atdA2 300 1058 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 ggggaattgt gagatatacc ctcggcgcgc taatcgtatt gagcggataa tggcagatct gctctgaaga actccgactg atggctttgt gcttatttga tcggttgtca agtttaggtt ccagtgaaga ttggatctac cgtatgcagt ttctacgggt ctgagctgag acaagcaaat tgagcaataa gaaacctcag aaaccagcaa gaatttgctt caggcgttta cgcagtactg gatgaacctg tagtgaaaac aactcaccca aggccaggtt aatcgtcgtg tgtaacaagg actccggatg gcttattttt aggtacattg tatcaacggt atctcgataa aacctcttac tatcaacagg attcggcgca ttttgaggtg ggggtggtgc cttactatgt ggctgcaccg tgactcgcta agatttcctg ccgtttttcc tggtggcgaa tgcgctctcc gagcggataa atgggcagca ctgcaggtcg gtacacggcc caattcccca caattggata agagcgcttt ggaagttata tatcagtggt atttattcga gtcccttgct tggaactgat gcgagtgagg attactcgca cggtatccaa tcatctcgaa tggttatcag taattttcag ctagcataac gcatttgaga tagacataag tcgaatttct agggcaccaa ttgtaattca aatcgccagc gggggcgaag gggattggct ttcaccgtaa gtattcactc gtgaacacta agcattcatc ctttacggtc agcaactgac ggtatatcca ctcaaaaaat gtgccgatca gacaccagga aagtgcgtcg ctccagtggc gtaacggcaa tggcactgat gtgcgtcagc cgctcggtcg gaagatgcca ataggctccg acccgacagg tgttcctgcc caattcccct gccatcacca acaagcttgc gcataatcga tcttagtata tcggccggcc gattatcgag agtgcattca agtgagtact gcggtccata gtcgcacttg gagctcacgt cttattgaaa cgttctgatt ggacatccag gatggtaatt cctcctcaag aacttggtgg cccttggggc agcacacggt cggctattta gccattcatc taactgcctt ttaagcattc ggcatcagca aagttgtcca gagacgaaaa cacgccacat cagagcgatg tcccatatca aggcgggcaa tttaaaaagg tgaaatgcct gtgatttttt acgcccggta acgtctcatt tttatttatt ggtgatgctg ttctgtttct aagcaccgcc gagggtgtca agaatatgtg ttcgactgcg ggaagatact cccccctgac actataaaga tttcggttta gtagaaataa tcatcaccac ggccgcataa aattaatacg ttagttaagt acatgtctaa aagaaatggt cagacttaaa cagttaatgc agaaagaaaa gcggagaggt ttcaaaatgg gccatggaga caactgctgt agatcagtaa tgcaagaaga cgatacgtca gtgatgtata ctctaaacgg cacactgctt acgaccctgc cgcttattat aaaaaaatta tgccgacatg ccttgtcgcc tattggccac acatattctc cttgcgaata aaaacgtttc ccagctcacc gaatgtgaat ccgtaatatc caaaatgttc tctccatttt gtgatcttat ttcgccaaaa ctgcgaagtg ccaacttact atcagctgtc ggacatcagc gtgaagtgct atacaggata gcgagcggaa taacagggaa aagcatcacg taccaggcgt ccggtgtcat ttttgtttaa agccaggatc tgcttaagtc actcactata ataagaagga acgctttgca aaatgccttt taaaattatc tgataaagaa accaattgtt gggtttgaac acttaacaaa atgcgtcatt agaaattttt aaaaaccctg tgaggtacgc attagtgaaa acctaggctg gtcttgaggg ccggtagtca cctgaaccga cacttattca cgccccgccc gaagccatca ttgcgtataa gtttaaatca aataaaccct tatgtgtaga agtttgctca gtctttcatt aaaggccgga cagctgaacg tttacgatgc agcttcctta ttcattatgg gttggcccag atcttccgtc gatttagtgt cctcctgttc gctagcggag tcatgtggca tattccgctt atggcttacg gtgagagggc aaatctgacg ttcccctggc tccgctgtta ctttaataag cgaattcgag gaacagaaag ggggaattgt gatatacata ttattgtggt aaaactgaaa gataattacg aagttttctg ggcatatgct cctagtcgtg catgttggta agacgcccac gcggtggggc gagcaagact aggtttcatg gcgactctac ctgccaccgc gttttttgct ataaaccggt cgaccgggtc ggcgtagcac tgccactcat cagacggcat tatttgccca aaactggtga ttagggaaat aactgccgga tggaaaacgg gccatacgga taaaacttgt gtctggttat cattgggata gctcctgaaa tgaaagttgg ggcttcccgg acaggtattt atgatggtgt agctactgac tgtatactgg ggagaaaaaa cctcgctcac aacggggcgg cgcggcaaag ctcaaatcag ggctccctcg tggccgcgtt 197 Appendix A 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 tgtctcattc atgcacgaac tccaacccgg ggagttagtc ctgcgctcct aaaaccgccc acgatctcaa tatctcttca ttagtcatgc ggtcgagatc cccgctttcc gggagaggcg caacagctga ggtttgcccc gctgtcttcg ctcggtaatg gggaacgatg gtcgccttcc agccagacgc gtgacccaat aatactgttg ggcagcttcc gacgcgttgc taccatcgac aatttgcgac tttgcccgcc ttccactttt ggtctgataa caccaccctg ccattcgatg taatacgact cacgcctgac cccccgttca aaagacatgc ttgaagtcat ccaagccagt tgcaaggcgg gaagatcatc aatgtagcac cccgcgccca ccggtgccta agtcgggaaa gtttgcgtat ttgcccttca agcaggcgaa gtatcgtcgt gcgcgcattg ccctcattca cgttccgcta agacgcgccg gcgaccagat atgggtgtct acagcaatgg gcgagaagat accaccacgc ggcgcgtgca agttgttgtg tcccgcgttt gagacaccgg aattgactct gtgtccggga cactata actcagttcc gtccgaccgc aaaagcacca gcgccggtta tacctcggtt ttttttcgtt ttattaatca ctgaagtcag ccggaaggag atgagtgagc cctgtcgtgc tgggcgccag ccgcctggcc aatcctgttt atcccactac cgcccagcgc gcatttgcat tcggctgaat agacagaact gctccacgcc ggtcagagac catcctggtc tgtgcaccgc tggcacccag gggccagact ccacgcggtt tcgcagaaac catactctgc cttccgggcg tctcgacgct gggtaggcag tgcgccttat ctggcagcag aggctaaact caaagagttg ttcagagcaa gataaaatat ccccatacga ctgactgggt taacttacat cagctgcatt ggtggttttt ctgagagagt gatggtggtt cgagatgtcc catctgatcg ggtttgttga ttgattgcga taatgggccc cagtcgcgta atcaagaaat atccagcgga cgctttacag ttgatcggcg ggaggtggca gggaatgtaa gtggctggcc gacatcgtat ctatcatgcc ctcccttatg ttcgctccaa ccggtaacta ccactggtaa gaaaggacaa gtagctcaga gagattacgc ttctagattt tataagttgt tgaaggctct taattgcgtt aatgaatcgg cttttcacca tgcagcaagc aacggcggga gcaccaacgc ttggcaacca aaaccggaca gtgagatatt gctaacagcg ccgtcttcat aacgccggaa tagttaatga gcttcgacgc cgagatttaa acgccaatca ttcagctccg tggttcacca aacgttactg ataccgcgaa cgactcctgc gctggactgt tcgtcttgag ttgatttaga gttttggtga gaaccttcga gcagaccaaa cagtgcaatt aattctcatg caagggcatc gcgctcactg ccaacgcgcg gtgagacggg ggtccacgct tataacatga gcagcccgga gcatcgcagt tggcactcca tatgccagcc cgatttgctg gggagaaaat cattagtgca tcagcccact cgcttcgttc tcgccgcgac gcaacgactg ccatcgccgc cgcgggaaac gtttcacatt aggttttgcg attaggaaat // 198 Appendix A A.6 pET A4A5 sequence FEATURES lacI Location/Qualifiers complement(5449 6531) PBR322 origin 4255 4256 Amp resistance 2637 3494 T7 promoter 6922 6938 T7 promoter 214 230 atdA4 300 926 atdA5 940 1947 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 ggggaattgt gagatatacc ctcggcgcgc taatcgtatt gagcggataa tggcagatct aaattgttga attatttttg ttctgactaa ttctggagga aggccacata cgggttttgc aagtatgggc ttgaaagccg aggataatga cgcaaggcaa gttattgata ggtgtcttgg ggtcttttat atcacggtaa gttggcgact cgagattttg acagcgctaa tctataatat atacaattct attgatgatg gtagagaatt gctgggagtg actgtaaact attttaaacg tgtgggtctt ttggacgctt aaaaatatag tagcataacc ctatatccgg ggtggttacg tttcttccct gctcccttta gggtgatggt ggagtccacg ctcggtctat tgagctgatt tggcggcacg aaatgaagtt tgcttaatca gagcggataa atgggcagca ctgcaggtcg gtacacggcc caattcccca caattggata tcctagcgct gaatctttct agaagctcgg tggctgttac tgagtttcat ctattcgcag agtgccgggg agatggcaag tgagttgaag taattcattt agatagcgga ctgagcactc ttcggtctta agagagaaag ttatcgagac ttttttttgc atagacacaa ttcataaaga ggttagacga ctctagaggt ttttgatcga tttctgatga ttatgctaaa agataataaa gcatgtgtct ctgatgagga aaatatcctt ccttggggcc attggcgaat cgcagcgtga tcctttctcg gggttccgat tcacgtagtg ttctttaata tcttttgatt taacaaaaat atggcatgag ttaaatcaat gtgaggcacc caattcccct gccatcacca acaagcttgc gcataatcga tcttagtata tcggccggcc gttaacctcg aaccttggtg cttttggatc tggattcctg gatatacgca atacctgttt tcaagcgagg tctcaagttt ataaagatga tttctatagt aacgaaggag ccctggcaag ttccctatct gggaggcaga actcccccct aggaggtagt aaataggatt gttaaaagat tgaaaaaggt tgaatatttt gtctaaagtc taatggcgat tggcattaag agctggaatt gctggtgagt agacggctgg cgaccaataa tctaaacggg gggacgcgcc ccgctacact ccacgttcgc ttagtgcttt ggccatcgcc gtggactctt tataagggat ttaacgcgaa attatcaaaa ctaaagtata tatctcagcg ctagaaataa tcatcaccac ggccgcataa aattaatacg ttagttaagt acatgaataa cttattatca agcctgcgct agcagtgttt gcagtatgcc gactgaaaga caaaaactaa ggtttttggt taagtggttg aacagataaa tggtctaaaa tcgttttcat tatttaccaa tcgtcagctt gggtcgaact gctggcagtt ggtataactc aagttgtttt ctatgtttac attccaacct ttatgtggac ccacctggac acagttgaaa aacagcgtta aatgttccta ggagatgtaa atcttggcgt cctaggctgc tcttgagggg ctgtagcggc tgccagcgcc cggctttccc acggcacctc ctgatagacg gttccaaact tttgccgatt ttttaacaaa aggatcttca tatgagtaaa atctgtctat ttttgtttaa agccaggatc tgcttaagtc actcactata ataagaagga taataaagat agagattaag tgatcacaag tgatgagtgg ggcggcgtca tcgaattgta tcgcatcctt tagaacaagc gtatggttat tttaaacgac tgaatacatt ttgttctgaa ttaaaatccg cggccaatga ggttgtgtga tccacccgca ctgtaatatc atgctaactc aattcccgga ctatcgcgtt ctgccccctt tgataaccaa gctcagctga tgtgctccga gttcgtgctg ttctagaaag gtcgatctaa tgccaccgct ttttttgctg gcattaagcg ctagcgcccg cgtcaagctc gaccccaaaa gtttttcgcc ggaacaacac tcggcctatt atattaacgt cctagatcct cttggtctga ttcgttcatc ctttaagaag cgaattcgag gaacagaaag ggggaattgt gatatacata cttagtataa cagtactctg ataaatatgt ttaacactgt cctgccagcg aggctgcaaa ggggcgccag tttattgtgt gtaattatta tgtctttcgc aaaatttcga gccgttggac aactgaaaaa ggactttaaa caatgtaaag gaactgggat cattataaaa ctctgaaagt tcggcttgat tgagcaatat catgggaggt ggagtctttt aaaagatgtc agatgatttt tgctggtaat caatactgtt accgagatcg gagcaataac aaaggaggaa cggcgggtgt ctcctttcgc taaatcgggg aacttgatta ctttgacgtt tcaaccctat ggttaaaaaa ttacaatttc tttaaattaa cagttaccaa catagttgcc 199 Appendix A 2701 2761 2821 2881 2941 3001 3061 3121 3181 3241 3301 3361 3421 3481 3541 3601 3661 3721 3781 3841 3901 3961 4021 4081 4141 4201 4261 4321 4381 4441 4501 4561 4621 4681 4741 4801 4861 4921 4981 5041 5101 5161 5221 5281 5341 5401 5461 5521 5581 5641 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 tgactccccg gcaatgatac gccggaaggg aattgttgcc gccattgcta ggttcccaac tccttcggtc atggcagcac ggtgagtact ccggcgtcaa ggaaaacgtt atgtaaccca gggtgagcaa tgttgaatac tctcatgagc aaatccctta gatcttcttg cgctaccagc ctggcttcag accacttcaa tggctgctgc cggataaggc gaacgaccta ccgaagggag cgagggagct tctgacttga ccagcaacgc ttcctgcgtt ccgctcgccg gcctgatgcg cactctcagt ctacgtgact cgggcttgtc atgtgtcaga tcagcgtggt agtttctcca ttttcctgtt ataccgatga ttactggaac atcactcagg cagcagcatc tccagacttt gttttgcagc gtaaggcaac atgccccgcg gatcccggtg ttccagtcgg ggcggtttgc ctgattgccc ccccagcagg ttcggtatcg aatggcgcgc gatgccctca ttcccgttcc acgcagacgc caatgcgacc gttgatgggt ttccacagca ttgcgcgaga cgacaccacc cgacggcgcg cgccagttgt tttttcccgc tcgtgtagat cgcgagaccc ccgagcgcag gggaagctag caggcatcgt gatcaaggcg ctccgatcgt tgcataattc caaccaagtc tacgggataa cttcggggcg ctcgtgcacc aaacaggaag tcatactctt ggatacatat acgtgagttt agatcctttt ggtggtttgt cagagcgcag gaactctgta cagtggcgat gcagcggtcg caccgaactg aaaggcggac tccaggggga gcgtcgattt ggccttttta atcccctgat cagccgaacg gtattttctc acaatctgct gggtcatggc tgctcccggc ggttttcacc cgtgaagcga gaagcgttaa tggtcactga aacgagagag gttgtgaggg gtcaatgcca ctgcgatgca acgaaacacg agcagtcgct cccgccagcc cccaccggaa cctaatgagt gaaacctgtc gtattgggcg ttcaccgcct cgaaaatcct tcgtatccca attgcgccca ttcagcattt gctatcggct gccgagacag agatgctcca gtctggtcag atggcatcct agattgtgca acgctggcac tgcagggcca tgtgccacgc gttttcgcag aactacgata acgctcaccg aagtggtcct agtaagtagt ggtgtcacgc agttacatga tgtcagaagt tcttactgtc attctgagaa taccgcgcca aaaactctca caactgatct gcaaaatgcc cctttttcaa ttgaatgtat tcgttccact tttctgcgcg ttgccggatc ataccaaata gcaccgccta aagtcgtgtc ggctgaacgg agatacctac aggtatccgg aacgcctggt ttgtgatgct cggttcctgg tctgtggata accgagcgca cttacgcatc ctgatgccgc tgcgccccga atccgcttac gtcatcaccg ttcacagatg tgtctggctt tgcctccgtg gatgctcacg taaacaactg gcgcttcgtt gatccggaac gaaaccgaag tcacgttcgc tagccgggtc ggagctgact gagctaactt gtgccagctg ccagggtggt ggccctgaga gtttgatggt ctaccgagat gcgccatctg gcatggtttg gaatttgatt aacttaatgg cgcccagtcg agacatcaag ggtcatccag ccgccgcttt ccagttgatc gactggaggt ggttgggaat aaacgtggct cgggagggct gctccagatt gcaactttat tcgccagtta tcgtcgtttg tcccccatgt aagttggccg atgccatccg tagtgtatgc catagcagaa aggatcttac tcagcatctt gcaaaaaagg tcatgattga ttagaaaaat gagcgtcaga taatctgctg aagagctacc ctgtccttct catacctcgc ttaccgggtt ggggttcgtg agcgtgagct taagcggcag atctttatag cgtcaggggg ccttttgctg accgtattac gcgagtcagt tgtgcggtat atagttaagc cacccgccaa agacaagctg aaacgcgcga tctgcctgtt ctgataaagc taagggggat atacgggtta gcggtatgga aatacagatg ataatggtgc accattcatg tcgcgtatcg ctcaacgaca gggttgaagg acattaattg cattaatgaa ttttcttttc gagttgcagc ggttaacggc gtccgcacca atcgttggca ttgaaaaccg gcgagtgaga gcccgctaac cgtaccgtct aaataacgcc cggatagtta acaggcttcg ggcgcgagat ggcaacgcca gtaattcagc ggcctggttc taccatctgg tatcagcaat ccgcctccat atagtttgcg gtatggcttc tgtgcaaaaa cagtgttatc taagatgctt ggcgaccgag ctttaaaagt cgctgttgag ttactttcac gaataagggc agcatttatc aaacaaatag ccccgtagaa cttgcaaaca aactcttttt agtgtagccg tctgctaatc ggactcaaga cacacagccc atgagaaagc ggtcggaaca tcctgtcggg gcggagccta gccttttgct cgcctttgag gagcgaggaa ttcacaccgc cagtatacac cacccgctga tgaccgtctc ggcagctgcg catccgcgtc gggccatgtt ttctgttcat ctgatgatga tgcggcggga taggtgttcc agggcgctga ttgttgctca gtgattcatt ggagcacgat ctctcaaggg cgttgcgctc tcggccaacg accagtgaga aagcggtcca gggatataac acgcgcagcc accagcatcg gacatggcac tatttatgcc agcgcgattt tcatgggaga ggaacattag atgatcagcc acgccgcttc ttaatcgccg atcagcaacg tccgccatcg accacgcggg ccccagtgct aaaccagcca ccagtctatt caacgttgtt attcagctcc agcggttagc actcatggtt ttctgtgact ttgctcttgc gctcatcatt atccagttcg cagcgtttct gacacggaaa agggttattg gtcatgacca aagatcaaag aaaaaaccac ccgaaggtaa tagttaggcc ctgttaccag cgatagttac agcttggagc gccacgcttc ggagagcgca tttcgccacc tggaaaaacg cacatgttct tgagctgata gcggaagagc atatatggtg tccgctatcg cgcgccctga cgggagctgc gtaaagctca cagctcgttg aagggcggtt gggggtaatg acatgcccgg ccagagaaaa acagggtagc cttccgcgtt ggtcgcagac ctgctaacca catgctagtc catcggtcga actgcccgct cgcggggaga cgggcaacag cgctggtttg atgagctgtc cggactcggt cagtgggaac tccagtcgcc agccagccag gctggtgacc aaataatact tgcaggcagc cactgacgcg gttctaccat cgacaatttg actgtttgcc ccgcttccac aaacggtctg 200 Appendix A 6481 6541 6601 6661 6721 6781 6841 6901 ataagagaca cctgaattga gatggtgtcc gtagtaggtt cgcccaacag tgagcccgaa caaccgcacc tcgatctcga ccggcatact ctctcttccg gggatctcga gaggccgttg tcccccggcc gtggcgagcc tgtggcgccg tcccgcgaaa ctgcgacatc ggcgctatca cgctctccct agcaccgccg acggggcctg cgatcttccc gtgatgccgg ttaatacgac gtataacgtt tgccataccg tatgcgactc ccgcaaggaa ccaccatacc catcggtgat ccacgatgcg tcactata actggtttca cgaaaggttt ctgcattagg tggtgcatgc cacgccgaaa gtcggcgata tccggcgtag cattcaccac tgcgccattc aagcagccca aaggagatgg caagcgctca taggcgccag aggatcgaga // 201 [...]... specificity of aniline dioxygenase The lack of characterization of the structural determinant of the substrate specificity of AtdA limits its development as a biocatalyst for industrial applications Hence, elucidation of the molecular determinants of the substrate specificity of AtdA is first required before engineering of the enzyme to expand its substrate range 2 Introduction In addition to bioremediation applications, ... the understanding of the structural determinants of the substrate specificity of AtdA, and enhanced the substrate range and activity of AtdA, making it a better enzyme for bioremediation The 3-R21 mutant created also serves as a useful platform in the stepwise evolution strategy to engineer AtdA for carbazole denitrogenation application x List of Tables Table 2.1 Summary of biomolecular engineering. .. reaction of aniline and its homologues by AtdA 62 Figure 4.2 Calibration curve of ammonium concentration using the indophenol blue assay 72 Figure 4.3 (A) Formation of the active coupling intermediate of MBTH (B) Electrophilic substitution of the intermediate by aniline to form the colored compound 74 Figure 4.4 Absorbance spectrum of MBTH assay with aniline and a mixture of catechol and aniline 74 Figure... spectrum of Gibbs’ reagent with aniline and its homologues 80 xiii Figure 4.12 Rate of color formation of aniline and catechol when reacted with Gibbs’ reagent at pH 5.8 81 Figure 4.13 Absorbance of the products of Gibbs’ reagent and catechol -aniline mixtures with time 82 Figure 4.14 Absorbance of colored products from the reaction of Gibbs’ reagent with aniline- catechol and 2IPA-3IPC mixtures Figure 4.15... expense of its activity for aniline (AN) and 2,4-dimethylaniline (24DMA) This is the first study on the molecular determinants for substrate specificity of a five subunit Rieske -dioxygenase, AtdA, and it was shown that the α-subunit of the enzyme (AtdA3) indeed plays a part in controlling the substrate specificity and activity of the enzyme Using knowledge gained from these findings, saturation and random... denitrogenation of fossil fuels." Trends in Biotechnology 16 (9): 390-395 Bomhard, E M and B A Herbold (2005) "Genotoxic activities of aniline and its metabolites and their relationship to the carcinogenicity of aniline in the spleen of rats." Crit Rev Toxicol 35 (10): 783-835 Bugg, T D H and C J Winfield (1998) "Enzymatic cleavage of aromatic rings: mechanistic aspects of the catechol dioxygenases and later... enzyme and the V205A and I248L mutants 138 Table 6.4 500 MHz 1H-NMR data (TMS internal standard) for 24DMA dihydroxylation product 138 Table 7.1 Sequences of primers used in saturation mutagenesis which were changed for the second and third round of mutagenesis 152 Table 7.2 Conversion rate of aniline, 24DMA, and 2IPA by E coli JM109 expressing the AtdA mutants 1-K31 and 2-A21 160 Table 7.3 The number of. .. 3-R21 and its parent 2-A21 166 Figure 7.8 Location of V205A, I248L, and S404C mutation in the AtdA3 subunit 168 Figure7.9 Residue 404 and its neighboring residues in (A) mutant 2-A21 and (B) mutant 3-R21 169 Figure 7.10 Activities of WT, 1-K31, 2-A21, and 3-R21 for (A) AN, (B) 24DMA, and (C) 2IPA Figure 8.1 Schematic of the project objective and scope 172 180 xvi Nomenclature 1NDO Crystal structure of. .. napthalene dioxygenase from Pseudomonas sp strain NCIB 9816-4 1ULJ Crystal structure of biphenyl dioxygenase from Rhodococcus sp strain RHA1 1WQL Crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01 24DMA 2,4-Dimethylaniline 2ABPD 2-Aminobiphenyl-2,3-diol 2EA 2-Ethylaniline 2IPA 2-Isopropylaniline 2MA 2-Methylaniline 2SBA 2-Sec-butylaniline 2TBA 2-Tert-butylaniline 34DMA 3,4-Dimethylaniline... probe for the molecular determinants of its substrate specificity as well as its activity Using the insights gained from the characterization studies, biomolecular engineering techniques were then used to improve the activity of AtdA as well as to expand its substrate range for application in bioremediation and industrial applications The first part of the dissertation presents the development of the . ENGINEERING OF ANILINE DIOXYGENASE FOR BIOREMEDIATION AND INDUSTRIAL APPLICATIONS ANG EE LUI NATIONAL UNIVERSITY OF SINGAPORE & UNIVERSITY OF ILLINOIS. ENGINEERING OF ANILINE DIOXYGENASE FOR BIOREMEDIATION AND INDUSTRIAL APPLICATIONS ANG EE LUI B. Eng (Hons.), National University of Singapore A THESIS SUBMITTED FOR. activity of AtdA as well as to expand its substrate range for application in bioremediation and industrial applications. The first part of the dissertation presents the development of the tools