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1 2 Genetic Program Genetic Program Parameter 3 Genetic Program - PowerPoint PPT Presentation

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1 2 Genetic Program Genetic Program Parameter 3 Genetic Program Genetic Program 4 Softcoding Platform Softcoding Platform Faster More affordable More affordable More predictable 5 Modulating Softcoding Platform g Genetic

  1. 1

  2. 2 Genetic Program Genetic Program Parameter

  3. 3 Genetic Program Genetic Program

  4. 4 Softcoding Platform Softcoding Platform • Faster • More affordable More affordable • More predictable

  5. 5 Modulating Softcoding Platform g Genetic Circuits VS Electronic Circuits

  6. 6 RNA Controller Toolkit RNA Controller Toolkit RNA Controller = Toolkit Toolkit Ligand responsive riboswitch/ribozyme Ligand concentration Translation strength

  7. 7 Softcoding Platform

  8. 8 RBS Calculator RBS Calculator RBS RBS calculator l l = RBS RBS Calculator RBS sequence Translation strength

  9. 9 Softcoding Platform AND AND gate t Vi l Violacein synthetic i th ti Bistable switch i bl i h pathway

  10. 10 RNA Controller Toolkit

  11. 11 RNA Controller Toolkit Criteria Criteria Appropriate range of regulation Modularity Orthogonality Orthogonality

  12. 12 Outline Outline  Harvest from existing RNA controllers  Develop a rational and semi ‐ rational design of ribozyme

  13. Existing RNA Controllers 13 Actuator Aptamer Actuator Aptamer Thiamine Pyrophosphate (TPP) Thi i P h h t (TPP) Th Theophylline Regulated h lli R l t d Regulated Ribozyme Riboswitch Jörg S. Hartig et al. (2009). Artificial Ribozyme Switches Containing Natural Riboswitch Aptamer Domains. Angew. Chem. 121, 2753 ‐ Jörg S. Hartig et al. (2009). Artificial Ribozyme Switches Containing Natural Riboswitch Aptamer Domains. Angew. Chem. 121, 2753 2756 Wolfgang Hillen et al. (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo . Nucleic Acids Research , 32 , 1610 ‐ 1614

  14. Theophylline Regulated Riboswitch 14 Result Result

  15. TPP Regulated Ribozyme 15 construction eGFP eGFP GFP GFP GFP GFP RBS RBS RBS RBS STOP T7 TPP STOP T7 TPP GFP RBS pBAD pBAD STOP TPP TPP CI GFP RBS RBS STOP pBAD TPP

  16. TPP Regulated Ribozyme 16 Result GFP T7 eGFP GFP T7 GFP pBAD CI GFP pBAD

  17. A General Method for 17 RNA Controller Construction RNA Controller Construction Design Various aptamers Hammerhead ribozyme Linker domain Various aptamers Hammerhead ribozyme

  18. A General Method for 18 RNA Controller Construction RNA Controller Construction selection TetA +NiCl 2 , Library y OFF selection OFF l ti ‐ Ligand, gene repression +Ligand, gene activation + Tc + Tc ON selection Yohei Yokobayashi et al.(2009) An efficient platform for genetic selection and screening of gene switches in Escherichia coli. Nucleic Acids Research. 37:e39

  19. A General Method for 19 RNA Controller Construction RNA Controller Construction Mock selection result Selected Selected 4 /off ratio 3 Selected on/ 2 Control Control Selected Control 1 1 Contro 0 ol 10 ‐ 1 10 ‐ 2 dilution

  20. A General Method for 20 RNA Controller Construction RNA Controller Construction Activation Sequence(5’->3’) fold Wild TTCATA TTCATA aptamer)..TATGAA t ) TATGAA 2 5 2.5 type 1.1 CCCATA CCCATA aptamer)..TATATG aptamer)..TATATG 4 OFF ON 1.4 CAAATA…(aptamer)..TATCTT 2.5 2.1 CTGATA..(aptamer)..TATGTA 1.5 2.2 ATGATA..(aptamer)..TATTTA 1.5

  21. Tran nslatio on S treng gth 21

  22. 22 RBS Calculator

  23. 23 Translation th Strengt S Strength h S Δ G G on nslatio Particular Particular Tra Sequence Sequence Ligand Concentration

  24. Derivation of Δ G 24 A Thermodynamics based Model A Thermodynamics ‐ based Model     G G G : standby mRNA mRNA rRNA nergy s Free En Gibbs Reaction Process Salis, H.M., Mirsky, E.A., and Voigt, C.A.(2009). Automated design of synthetic ribosome binding sites to control protein expression. Nat.Biotech., 27 :946:952

  25. Derivation of Δ G 25 A Thermodynamics based Model A Thermodynamics ‐ based Model       G G G G G : standby spacing mRNA mRNA rRNA start nergy s Free En Gibbs Reaction Process Salis, H.M., Mirsky, E.A., and Voigt, C.A.(2009). Automated design of synthetic ribosome binding sites to control protein expression. Nat.Biotech., 27 :946:952

  26. The RBS Calculator 26   G  G tot          G G G G G : m RNA m RNA rRNA standby spacing start Ke     G Translation Rate tot Translation Rate Salis, H.M., Mirsky, E.A., and Voigt, C.A.(2009). Automated design of synthetic ribosome binding sites to control protein expression. Nat.Biotech., 27 :946:952

  27. The RBS Calculator 27 Verification of Accuracy Verification of Accuracy RBS AUG Green Fluorescent Protein Output Fluorescence Measurement p

  28. The RBS Calculator 28 Verification of Accuracy Verification of Accuracy

  29. The RBS Calculator 29 → Forward Engineering → Forward Engineering Output Fluorescence Output Fluorescence (Translation Rate) RBS AUG Green Fluorescent Protein

  30. The RBS Calculator 30 → F → Forward Engineering d E i i rna1995, 51nt

  31. 31 AND Gate Module

  32. Architecture of AND gate 32 Input1 Input2 Input 1 supD T7 GFP UAG UAG Input 2 UAG * * | | T7ptag T7ptag RBS RBS

  33. Phase diagram 33 * * | | | | T7 t T7ptag RBS RBS

  34. 34 Step 1 TPP Hammerhead a e ead Ribozyme * * * * | | T7ptag T7ptag RBS RBS

  35. 35 Result of step 1

  36. 36 Step 2 p * * | | | | T7ptag RBS

  37. 37 Result of step 2 ∆ G= 1.33 kJ/mol ∆ G= -0.4873 kJ/mol RBS: RBS: ACACAGGAC GGAGUGG ACACAGGAC GGAGUGG

  38. 38 Bistable Switch Module Bistable Switch Module

  39. 39 Construction of Bistable Switch Construction of Bistable Switch CI CI 434 mRFP mRFP eGFP eGFP P RM P R

  40. 40 Softcoding of Bistable Switch CI mRFP mRFP CI 434 CI 434 eGFP eGFP P RM P R TPP hammerhead ribozyme TPP hammerhead ribozyme +TPP No TPP dG=4.6kJ/mol High TPP conc. dG=7.0kJ/mol Translation strength of CI434

  41. Stochastic Model of Bistable Switch 41

  42. Stochastic Model of Bistable Switch 42 Fit with Softcoding Result

  43. Prediction of a RBS sequence 43 No TPP ： dG=4 6kJ/mol No TPP ： dG=4.6kJ/mol High TPP conc. ： dG=7.0kJ/mol dG=5.3kJ/mol RBS calculator RBS calculator RBS: CGAGACC RBS: CGAGACC

  44. 44 Fi Fine ‐ tuning i bi biosynthetic pathway th ti th

  45. Violacein pathway 45 deoxychromoviridans H H N N N N H H N N H H

  46. Violacein pathway 46 deoxychromoviridans H H N N N N H H N N H H

  47. Design 47 IPA imine L ‐ tryptophan VioA VioB VioB IPA imine dimer dimer VioE VioE P Prodeoxyviolacein d i l i VioD VioD Proviolacein Deoxy ‐ chromoviridans chromoviridans

  48. Design 48 IPA imine dimer dimer VioE Prodeoxyviolacein VioD Proviolacein Deoxy ‐ chromoviridans h i id

  49. Fine ‐ tuning pathway 49 Translation Strength control violacein violacein violacein violacein

  50. Fine ‐ tuning pathway 50 Solvent ‐ TPP TPP 10nM TPP 100nM TPP 1  M TPP 10  M

  51. Fine ‐ tuning pathway 51

  52. 52 Summary

  53. 53 Idea Summary Idea Summary Soft coding Soft-coding Ligand Conc. Translation Strength RBS Sequence

  54. 54 Achievements Achievements • RNA controllers & RBS calculator – New ribozymes with different aptamers – Improved existing ribozymes – Improved Reverse translation strength prediction Improved Reverse translation strength prediction • Three applications – AND gate AND t – Bistable switch – Violacein bio ‐ synthesis

  55. 55 Achievements Achievements • RNA controllers & RBS calculator – New ribozymes with different aptamers – Improved existing ribozymes – Improved Reverse translation strength prediction Improved Reverse translation strength prediction • Three applications – AND gate AND t – Bistable switch – Violacein bio ‐ synthesis

  56. 56 RNA Controllers RNA Controllers

  57. 57 Achievements Achievements • RNA controllers & RBS calculator ✔ New ribozymes with different aptamers ✔ Improved existing ribozymes – Improved Reverse translation strength prediction Improved Reverse translation strength prediction • Three applications – AND gate AND t – Bistable switch – Violacein bio ‐ synthesis

  58. 58 RBS Calculator RBS Calculator RBS RBS AUG AUG Green Fluorescent Protein Green Fluorescent Protein

  59. 59 Achievements Achievements ✔ RNA controllers & RBS calculator ✔ New ribozymes with different aptamers ✔ Improved existing ribozymes ✔ Improved Reverse translation strength prediction ✔ Improved Reverse translation strength prediction • Three applications – AND gate AND t – Bistable switch – Violacein bio ‐ synthesis

  60. 60 Applications Applications CI mRFP CI 434 eGFP P RM P R

  61. 61 Prospect & future plans Prospect & future plans • New generation – Quantitative Q – Predictable – Fast Fast • Improvement – Precise – General General

  62. 62 Parts Parts • Favorite Parts Favorite Parts BBa_K598000 TPP Down ‐ regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015 BBa_K598001 Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator BBa_K598002 Bistable Switch Mutant 68 • Parts Sandbox BBa K598003 BBa K598027 BBa_K598003 ‐‐ BBa_K598027

  63. 63 Human Practice Human Practice

  64. 64 Collaboration Collaboration

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