Q1: Find the secret message in the sentences. Q2: Can I delete a w letter in these sentences?

Transcription

1 Q1: Find the secret message in the sentences. Why is DNA necessary? Life is very special and cells are great things. Significantly advanced life science is easily mistaken as magic. Q2: Can I delete a w letter in these sentences?

2 Session 4:Synthetic/constructive experiment of life and evolution of genome Daisuke Kiga ELSI / Dept of computational intelligence and systems science, Tokyo Institute of Technology Back ground: biochemistry Why 4 nucleotide and 20 amino acids? For resurrection of proteins before commonotes, engineered genetic code less than 20 amino acids is important Previous works: engineering of genetic code xanthine-biniding aptamer autonomous DNA computing, artificial genetic network in living cell

3 Acknoledgements 遺伝暗号の改変 21 番目のアミノ酸 理研 SSBC ( 横山グループ ) 19 アミノ酸遺伝暗号 木賀研 内山正彦 (NEDO 研究員 ) 小林晃大 網蔵和晃 無細胞タンパク合成 木川隆則 アミノ酸組成分析 理研堂前直 中山洋 益田晶子 質量分析 東大福沢世傑 児玉公一郎 結晶構造解析 東大荒磯裕平 濡木理 NEDO 産業技術助成事業 ( ) JSPS 科研費若手 (A) ( ), ( ) JSPS 科研費挑戦的萌芽 ( ) 人工遺伝子ネットワーク 東工大木賀研 鮎川翔太郎 畑敬士 石松愛 東工大山村雅幸 東大陶山研 陶山明 庄田耕一郎 瀧ノ上正浩 李泳薫 東大萩谷昌己 JSPS 科研費特定 分子プログラミング ( ) JSPS 科研費若手 (B) ( ) JST 先端計測 ( ) JSPS 科研費挑戦的萌芽 ( ) ( ) JST さきがけ ( ) JSPS 科研費若手 (A) ( ) JSPS 科研費新学術領域 合成生物学 計画研究 ( ) 赤字 : 研究代表

4 contents Synthetic/Constructive approach in life science proof by synthesis construction of another life Simplified genetic code for reconstitution of ancient protein code and protein with less than 20 amino acids How to engineer genetic code detailed results

5 contents Synthetic/Constructive approach in life science proof by synthesis construction of another life Simplified genetic code for reconstitution of ancient protein code and protein with less than 20 amino acids How to engineer genetic code

6 Understanding of System analytical approach! constructive approach! What I cannot create, I do not understand. -R. Feynman

7 Life is a multi layer system analytical approach! atom building block bio macromolecule subsystem of cell cell tissue individual Carbon Oxygen Nitrogen nucleotide amino acid lipid DNA RNA protein gene network protein synthesis Photosynthesis vesicle Recent Developments Comprehensive analysis: genome, proteome. Preparation of biomolecules

8 Developments in preparation of biomolecules Moor s Low in DNA synthesis: exponential growth DNA length DNA length Human genome length year artificial DNA Kiga and Yamamura, New Generation Computing, with human genome length!? 26 (2008), p Preparation of proteins Protein 3000: Structural genomics

9 Natural / unnatural combinations can be realized analytical approach! atom building block bio macromolecule subsystem of cell cell tissue individual Carbon Oxygen Nitrogen nucleotide amino acid lipid DNA RNA protein gene network protein synthesis Photosynthesis vesicle constructive approach! Synthetic Biology

10 President Tetsuya Yomo President Daisuke Kiga synthetic molecular system Mix proteins and DNA/RNA aptazyme system In vitro protein production DNA computation /molecular programming Can we synthesize a cell? ChemComm 2012 cover figure Japanese Society for Cell Synthesis Research ~μm

11 Objectives of Synthetic biology Engineering protein engineering metabolic engineering tissue engineering Science washing-powder Drug, Fuel, Fine chemical combination of cells

12 Objectives of Synthetic biology Engineering Process for Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, Ribosome PURE Translation system Regulatory circuit robust system Construction of another life

13 Objectives of Synthetic biology Engineering Waddington 1957 one phenotype multi phenotypes ips S Yamanaka Nature (2009) Natural Complicated system Epigenetic landscape fertilized egg differentiated cells Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, 13 Ribosome Process for robust system PURE Translation system Regulatory circuit Construction of another life Molecular Interaction Network in Human Organogenesis Fang et al, Dev Cell 2010

14 Objectives of Synthetic biology Kiga Lab PNAS (2011) vol108, Waddington 1957 one phenotype multi phenotypes ips High state H H H Cell state LLLLL L L Low High Low state concentration of communication molecule Synthetic Simple system in living cell/test tube Science Natural Complicated system Epigenetic landscape fertilized egg dyi = d t 1+ dxi = d t 1+ dz Ntot Reconstitution: dt N proof-by-synthesis Wet experiment & Modeling ATPase, RNA Pol, Ribosome PURE Translation system y nx i x ( x K ) Process for robust system nz z K + z ny x i ( y K ) Regulatory circuit Construction of another life α α i = λ x d z x nz z i y d x z nz d y y i differentiated cells 14 Molecular Interaction Network in Human Organogenesis Fang et al, Dev Cell 2010

15 Objectives of Synthetic biology Engineering protein engineering metabolic engineering tissue engineering Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, Ribosome PURE Translation system Regulatory circuit Construction of another life other natural systems made of natural parts what life could be in early earth or exoplanets

16 contents Synthetic/Constructive approach in life science Simplified genetic code for reconstitution of ancient protein code and protein with less than 20 amino acids How to engineer genetic code

17 Why 4 nucleotide and 20 amino acids?

18 Only one solution for one function? many alternative forms

19 Universal property in Present Life is Important for Life? Replication, Metabolism, Containment Carbon-based polymer L-amino acids, D-ribose Succession of DNA through generations DNA=Blue Print, Protein=Functional Molecule DNA (4 nucleotides) transcription mrna (4 nucleotides) translation Protein (20 amino acids) G A A C U C A G U A U A (E) (L) (S) (I) Glu Leu Ser Ile U A G stop

20 UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Leu Phe Leu Ile Met Val Ser Pro Thr Ala Tyr Stop Stop His Gln Asn Lys Asp Glu Cys Stop Trp Arg Ser Arg Gly from E. coli to Human The Universal genetic code

21 Sequence variety for a small protein (200 amino acids length) atoms in the Universe ~10 80 age of the Universe (sec) Nature creates little part of What life could be

22 Is set of natural 20 amino acids the only solution? The Univerasl Genetic Code UUU UUC UUA UUG Phe Leu UCU UCC UCA UCG Ser UAU UAC UAA UAG Tyr Stop Stop UGU UGC UGA UGG Cys Stop Trp CUU CUC CUA CUG Leu CCU CCC CCA CCG Pro CAU CAC CAA CAG His Gln CGU CGC CGA CGG Arg AUU AUC AUA AUG Ile Met ACU ACC ACA ACG Thr AAU AAC AAA AAG Asn Lys AGU AGC AGA AGG Ser Arg GUU GUC GUA GUG Val GCU GCC GCA GCG Ala GAU GAC GAA GAG Asp Glu GGU GGC GGA GGG Gly ~2005: all of the natural codes contains 20 aa.

23 UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Leu Leu Ile Met Val Why 20 amino acids in the Universal code? The universal genetic code UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala can we construct other codes? UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Tyr Stop Stop His Gln Asn Lys Asp Glu 20 UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Cys Stop Trp Arg Ser Arg Gly 21 UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Expanded genetic code Phe Leu Leu Ile Met Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Tyr Stop 21st aa His Gln Asn Lys Asp Glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Simplified genetic code Cys Stop Trp Arg Ser Arg Gly 3-iodo tyrosine Kiga et al., PNAS any mrna produces protein w/o Trp Kiga Lab Nucleic Acids Research 2012, featured article

24 synthetic molecular system Mix proteins and DNA/RNA Subsystem of early life Present life: 20 amino acid Early life : Less than 20 /Phylogenic analysis of components for protein production shows possibility of organisms with less than 20 AA /Chemical evolution experiments ~μm can produce not all 20 amino acids (exlude non-canonical amino acids /accept contamination of non-canonical AA) Can proteins with less than 20 amino acids provide activities for life system?

25 Artificial evolution of protein initial library mutation selection amplification Mutations are introduced to nucleotide sequence on DNA, not amino acid sequence on protein Can proteins with less than 20 amino acids provide activities for life system?

26 Artificial evolution of protein with less than 20 amino acids amino acids to be removed Previous strategy w/ universal code wild type Our strategy w/ simplified code wild type have to pick up Low activity mutant Mutant with no activity Low activity mutant Mutant with no activity mutagenesis mutagenesis High activity mutant High activity mutant Cannot use evolutionary process mutations on DNA restore codons for amino acids to be removed w/ simplified genetic code Can use evolutionary process amino acids to be removed never appear even with mutations on DNA

27 contents Synthetic/Constructive approach in life science Simplified genetic code for reconstitution of ancient protein code and protein with less than 20 amino acids How to engineer genetic code detailed results

28 UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Leu Phe Leu Ile Met Val Ser Pro Thr Ala Tyr Stop Stop His Gln Asn Lys Asp Glu Cys Stop Trp Arg Ser Arg Gly from E. coli to Human The Universal genetic code

29 Mechanism of the genetic code Amino acid codon

30 Mechanism of the genetic code Amino acid codon trna: adaptor Amino acid codon anticodon mrna (Blue Print)

31 echanism of the genetic code Aminoacyl-tRNA synthetase (aars) attaches amino acid to trna anticodon amino acid

32 Mechanism of the genetic code Natural 20 exclusive groups of amino acid, enzyme and trnas UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG The Univerasl Genetic Code Phe Leu Leu Ile Met UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG Ser Pro Thr UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG Tyr Stop Stop His Gln Asn Lys UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG Cys Stop Trp Arg Ser Arg amino acid Ala alars (enzyme) Thr trna trna Ala 1B trna 2 Ala trna 1,3 Thr trna 2 Thr trna Thr 4 ThrRS (enzyme) codon GCU GCC GCA GCG ACU ACC ACA ACG GUU GUC GUA GUG Val GCU GCC GCA GCG Ala GAU GAC GAA GAG Asp Glu GGU GGC GGA GGG Gly Tyr trna Tyr 1 trna Tyr 2 TyrRS (enzyme) UAU UAC

33 Genetic code with 21 amino acids by creation of additional group UUU UUC UUA UUG CUU CUC CUA CUG an expanded genetic code Phe Leu Leu UCU UCC UCA UCG CCU CCC CCA CCG Ser Pro UAU UAC UAA UAG CAU CAC CAA CAG Tyr Stop 21st aa His Gln UGU UGC UGA UGG CGU CGC CGA CGG Cys Stop Trp Arg amino acid Ala alars (enzyme) Thr trna trna Ala 1B trna 2 Ala trna 1,3 Thr trna 2 Thr trna Thr 4 ThrRS (enzyme) codon GCU GCC GCA GCG ACU ACC ACA ACG AUU AUC AUA AUG GUU GUC GUA GUG Ile Met Val ACU ACC ACA ACG GCU GCC GCA GCG Thr Ala AAU AAC AAA AAG GAU GAC GAA GAG Asn Lys Asp Glu AGU AGC AGA AGG GGU GGC GGA GGG Ser Arg Gly Tyr TyrRS (enzyme) 21st aa trna Tyr 1 trna Tyr 2 trna new UAU UAC UAG aars mutant enzyme Kiga, et al. PNAS (2002) new group without crosstalk

34 Expansion of genetic code system by addition of engineered components for the 21st amino acid 20 amino acid addition of engineered components new AA new parts for 21 st AA cell extract wt trna for 20 AA wt enzyme for 20 Kiga, et al. PNAS (2002)

35 Design of simplified genetic code in vitro Reassign Trp codon to Ala AlaRS Ala trna Ala Ala codon AlaRS trna Ala variant Trp trna Trp Trp codon(ugg) TrpRS trna C C A Ala AlaRS doesn t recognize the anticodon loop to attach Ala. Ala anticodon U G C Trp anticodon C C A 35

36 Results-I Sense codon suppression by trna Ala variant 14 C-labeled protein. AlaRS Ala trna Ala Ala codon AlaRS trna Ala variant trna Ala variant (µm) Trp Trp-SA Lane Trp trna Trp TrpRS Trp codon (UGG) Ala, not Trp, is likely to be inserted for UGG codons. Next: amino acid composition analysis 36

37 Results-I Synthesized protein did not contain Trp. Absorbance (A.U.) 3 2 Trp Retention Time (min) Wild type mrna mrna variant (UGG GCA) Wild type mrna Universal code Simplified code Trp codon Ala Protein: MBP with 8 Trp 37

38 Results-I Ala was increased instead of Trp disappearance. Amino acid composition analysis showed Ala and other amino acid s content. The increased amount of Ala was estimated as 8 residues, which is equivalent to the number of UGG codons in the MBP-WT mrna. The result strongly indicates that the trna variant introduced Ala to the UGG codon. 38

39 Results-I Simplified genetic code Trp あ r Ala Can produce active protein Ala Fluorescence (A.U.) GFP G FP U G G G FP U G G A110UGG mrna protein:gfp Natural Universal code (20 amino acid, UGG:Trp) Universal (U G G :T rp) Sm plified (U G G :A la ) Engineered Simplified code (19 amino acid, UGG:Ala) Gene specific for simplified genetic code OK??? 9801 barrier against horizontal transfer

40 Results-II trna Generality of simplified genetic codes Lys Ser Cys Ser Lys + trna Cys + Protein :St.Av. Protein: Ras Asn Ala trna Asn + Trp Ala trna Trp + Protein: Ras Protein :St.Av. Cys Ala, Asn Ser, Trp Ser Thr Ser.

41 Results-II The simplified genetic code comprising 16 amino acids Translation reaction with the trna Ser variants in the E.coli cell extract lacking specific amino acids. trna variant amino acids + aa-sas Lane Ser Ser Ser Ser trna variant : UGU/UGC:Ser, UGG:Ser, UAU/UAC:Ser, AAU/AAC:Ser 4amino acids : Cys, Trp, Tyr, Asn aa-sas : Cys-SA, Trp-SA, Tyr-SA, Asn-SA 41

42 For resurrection of proteins before commonotes, engineered genetic code less than 20 amino acids is important Low contents Synthetic/Constructive approach in life science proof by synthesis construction of another life Process for robust system High state H H H Cell state LLLLL L L High Low state concentration of communication molecule Simplified genetic code artificial evolution for reconstitution of ancient protein code and protein with less than 20 amino acids How to engineer genetic code Gene specific for simplified genetic code 16 aa genetic code, and so on

43 Q1: Find the secret message in the sentences. Why is DNA necessary? Life is very special and cells are great things. Significantly advanced life science is easily mistaken as magic. Q2: Can I delete the w letter in these sentences? The above sentences use 20 of 26 letters in English alphabet How about expression in German, Spanish, Japanese and so on?

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46 Simplification of the code: Construction of genetic codes construction of genetic code with 19 amino acids with 19 amino acids UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Leu Leu Ile Met Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Lys Tyr Stop Stop His Gln Asn Asp Glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Cys Stop Trp Arg Ser Arg Gly amino acid Lys LysRS AlaRS Ala Tyr TyrRS trna trna Lys trna Ala 1B trna 2 Ala trna Tyr 1 trna Tyr 2 codon AAA AAG GCU GCC GCA GCG UAU UAC Removal of an amino acids Unassigned codon

47 Simplification of the code: Construction of genetic codes construction of genetic code with 19 with amino 19 acids amino acids amino acid trna codon UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Phe Leu Leu Ile Met Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG Lys Tyr Stop Stop His Gln Asn Ala Asp Glu UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG Cys Stop Trp Arg Ser Arg Gly Lys LysRS AlaRS Ala Tyr TyrRS trna Lys trna Ala x trna Ala 1B trna 2 Ala trna Tyr 1 trna Tyr 2 AAA AAG GCU GCC GCA GCG UAU UAC Removal of an amino acids Addition of trna Ala/Ser mutant Unassigned codon Codon Reassignment

48 Simplification of the code: Codon reassignment by trna Ala variant trna Ala mutant Lys codon AAA/G Ala Lys + Lys-SA + laneno amino acid trna codon Lys LysRS trna Lys AAA AAG AlaRS Ala Tyr TyrRS trna Ala x trna Ala 1B trna 2 Ala trna Tyr 1 trna Tyr 2 GCU GCC GCA GCG UAU UAC

49 Simplification of the code: Amino acid composition analysis w/ RIKEN Dohmae group AAA/AAG codons are reassigned from Lysine(K) to Alanine(A) 25 % amino acid R H S G T P A D E Y V M K I + + N Q L F Universal code wt mrna (Theoretical value) wt mrna mrna variant (AAA/AAG GCA) Simplified code wt mrna

50 Simplification of the code: Generality of the method Lys Ser Cys Ser trna Lys + trna Cys + Protein :St.Av. Protein: Ras Asn Ala trna Asn + Trp Ala trna Trp + Protein: Ras Protein :St.Av. Cys Ala, Asn Ser, Trp Ser Thr Ser.

51 Simplification of the code: Simplified genetic code can produce active protein Gene specific for simplified genetic code Ala Fluorescence (A.U.) Universal (U G G :T rp) Sm plified (U G G :A la ) 0 GFP G FP U G G G FP U G G A110UGG OK?????? OK

52 Simplification of the code: Gene specific for simplified genetic code Fluorescence (A.U.) Universal (U G G :T rp) 0 GFP G FP U G G G FP U G G A110UGG codon 57 amino acid required at 57 amino acid programmed at 57 codon 110 amino acid required at 110 mino acid programmed at 110 UGG UUU UUU Trp Phe Phe Trp Phe Phe GCU GCU UGG Ala Ala Ala Ala Ala Trp

53 Simplification of the code: Gene specific for simplified genetic code Fluorescence (A.U.) Universal (U G G :T rp) 0 GFP G FP U G G G FP U G G A110UGG codon 57 amino acid required at 57 amino acid programmed at 57 codon 110 amino acid required at 110 mino acid programmed at 110 UGG UUU UUU Trp Phe Phe Trp Phe Phe GCU GCU UGG Ala Ala Ala Ala Ala Trp

54 Simplification of the code: Gene specific for simplified genetic code Fluorescence (A.U.) Universal (U G G :T rp) Sm plified (U G G :A la ) codon 57 amino acid required at 57 amino acid programmed at 57 codon 110 amino acid required at 110 mino acid programmed at GFP G FP U G G G FP U G G A110UGG UGG UUU UUU Trp Phe Phe Trp Ala Phe Phe Phe Phe GCU GCU UGG Ala Ala Ala Ala Ala Ala Ala Trp Ala

55 Simplification of the code: Gene specific for simplified genetic code Fluorescence (A.U.) Universal (U G G :T rp) Sm plified (U G G :A la ) codon 57 amino acid required at 57 amino acid programmed at 57 codon 110 amino acid required at 110 mino acid programmed at GFP G FP U G G G FP U G G A110UGG UGG UUU UUU Trp Phe Phe Trp Ala Phe Phe Phe Phe GCU GCU UGG Ala Ala Ala Ala Ala Ala Ala Trp Ala

56 Simplification of the code: Genetic code with 18 amino acids trna (Lys->Ala) trna (Cys->Ser) Lys Cys + + Lane No 1 Ser Ras Ala

57 Genetic Code: Conclusion The universal number of amino acids in the genetic code can be changed. Future works Protein with less than 20 amino acids evolution under different genetic codes Screening System Cell with less than 20 amino acids? have to construct artificial cell from scratch

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59 Number of amino acids and aminoacyl trna synthetases Canonical case: 20 aars :20 amino acids 19 aars : 20(=19+1) AA Organisms without Glutaminyl trna synthetase transfer of amino group to Glu on trna 20 aars : 20 AA + additional AA Selenocysteine incorporation transfer of selenium to Ser on trna 21 aars : 20 AA + additional AA Pyrrolysine incorporation (in nature) Iodotyrosine incorporation (engineered) 19 aars : 19 amino acids In a past form of life, less than 20 amino acids would be assigned in a genetic code

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61 Objectives of Synthetic biology Engineering protein engineering metabolic engineering tissue engineering Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, Ribosome PURE Translation system Regulatory circuit Construction of another life We still cannot construct cell w/ 20 aa

62 Japanese Society for Cell Synthesis Research President 2010 Shoji Takeuchi President 2012 Daisuke Kiga

63 Kiga Shimizu T. Ueda Japanese Society for Cell Umeno Protein synthesis Suga Information Processing Kobayashi Synthesis Research Gene Network Iwasaki H.R.Ueda Yoshizawa Kato Cell synthesis Itaya Genome Engineering Compartment. Interactions w/ Society Hashimoto Observation Takeuchi Yomo Toyoda Noji Suzuki Tabata Hibino

64 Objectives of Synthetic biology dx dt Engineering Protein engineering i = 1+ metabolic engineering tissue engineering α dy α i y = dt 1+ xi K x dz Ntot = λ x dt N ( ) Kiga Lab PNAS (2011) vol108, ( ) ny x i y K i x i y nx d z d K nz z z z x nz + z High state nz H H H d y Cell state y i LLLLL L L Low High Low state Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, Ribosome concentration of communication molecule PURE Translation system Regulatory circuit

65 Construction of Artificial Genetic Circuit by Copy-and-Paste from DNA sequence of Organisms Genome Information on Web Design of DNA sequence

66 Developments in preparation of biomolecules Moor s Low in DNA synthesis: exponential growth DNA length DNA length Human genome length year Kiga and Yamamura, New Generation Computing, 26 (2008), p biological synthesis:copy template chemical synthesis:any sequence artificial DNA with human genome length!? Preparation of proteins

67 Construction of Artificial Genetic Circuit by Copy-and-Paste from DNA sequence of Organisms have to design regulatory program Genome Information on Web Design of DNA sequence DNA synthesis Introduction of DNA to cell or artificial vesicle gene=regulatory+protein coding ~μm

68 Programming in computer and life computer Fix 0-1 state in a bit High energy consumption life Low energy consumption unable to fix its state 10molecules 20molecules µm size Reaction in tiny space containing a small number of molecules fluctuation in number 9 molecules? 10 molecules? 11 molecules?

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70 Programming in computer and life computer Fix 0-1 state in a bit High energy consumption life Low energy consumption unable to fix its state 10molecules 20molecules µm size Reaction in tiny space containing a small number of molecules fluctuation in number 9 molecules? 10 molecules? 11 molecules?

71 Kiga Shimizu T. Ueda Japanese Society for Cell Umeno Protein synthesis Suga Information Processing Kobayashi Synthesis Research Gene Network Iwasaki H.R.Ueda Yoshizawa Kato Cell synthesis Itaya Genome Engineering Compartment. Interactions w/ Society Hashimoto Observation Takeuchi Yomo Toyoda Noji Suzuki Tabata Hibino

72 Objectives of Synthetic biology Engineering one phenotype multi phenotypes Epigenetic landscape ips fertilized egg Science Reconstitution: proof-by-synthesis ATPase, RNA Pol, Ribosome PURE Translation system Regulatory circuit Construction of another life S Yamanaka Nature (2009) differentiated cells Molecular Interaction Network in Human Organogenesis Fang et al, Dev Cell 2010