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 - - PowerPoint PPT Presentation

May 01, 2023 427 likes •1.13k views

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

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SLIDE 1

1

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SLIDE 2

Genetic Program

2

Genetic Program

Parameter

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SLIDE 3

Genetic Program

3

Genetic Program

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SLIDE 4

Softcoding Platform

4

Softcoding Platform

  • Faster
  • More affordable

More affordable

  • More predictable
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SLIDE 5

Modulating

Softcoding Platform

5

Genetic Circuits VS Electronic Circuits

g

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SLIDE 6

RNA Controller Toolkit

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RNA Controller Toolkit

RNA Controller Toolkit

=

Toolkit Ligand responsive riboswitch/ribozyme Ligand concentration Translation strength

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SLIDE 7

Softcoding Platform

7

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SLIDE 8

RBS Calculator RBS l l

8

RBS Calculator RBS calculator

RBS

=

RBS Calculator

RBS sequence Translation strength

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SLIDE 9

9

Softcoding Platform

AND t i bl i h Vi l i th ti AND gate Bistable switch Violacein synthetic pathway

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SLIDE 10

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RNA Controller Toolkit

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SLIDE 11

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RNA Controller Toolkit

Criteria

Appropriate range of regulation

Criteria

Modularity Orthogonality Orthogonality

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SLIDE 12

Outline

12

Outline

 Harvest from existing RNA controllers

 Develop a rational and

semi‐rational design of ribozyme

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SLIDE 13

Existing RNA Controllers

13

Aptamer Actuator Actuator Thi i P h h t (TPP) Th h lli R l t d Aptamer Thiamine Pyrophosphate (TPP) Regulated Ribozyme Theophylline Regulated 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

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SLIDE 14

Result

Theophylline Regulated Riboswitch

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Result

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SLIDE 15

TPP Regulated Ribozyme

15

construction

RBS

GFP

RBS

eGFP GFP

RBS

GFP

T7

TPP

STOP

T7

RBS

TPP

STOP eGFP GFP

RBS

pBAD

TPP

STOP GFP

pBAD

TPP

RBS

CI GFP

RBS

TPP

STOP

pBAD

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SLIDE 16

TPP Regulated Ribozyme

16

Result

GFP

T7 T7

eGFP GFP

pBAD

GFP

pBAD

CI GFP

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SLIDE 17

A General Method for RNA Controller Construction

17

RNA Controller Construction

Design Hammerhead ribozyme Various aptamers

Linker domain

Hammerhead ribozyme Various aptamers

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SLIDE 18

A General Method for RNA Controller Construction

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RNA Controller Construction

selection

+NiCl2,

OFF l ti

Library

TetA

OFF selection

‐Ligand, gene repression

y

+ Tc +Ligand, gene activation + 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

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SLIDE 19

A General Method for RNA Controller Construction

19

RNA Controller Construction

Selected

Mock selection result

Selected

4

/off ratio

3

Selected Selected Control

  • n/

2 1

Control Control Contro

1

  • l

10‐1 10‐2 dilution

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SLIDE 20

A General Method for RNA Controller Construction

20

RNA Controller Construction

Sequence(5’->3’) Activation fold Wild TTCATA

t ) TATGAA

2 5 type

TTCATA aptamer)..TATGAA

2.5 1.1

CCCATA aptamer)..TATATG

4

CCCATA aptamer)..TATATG

1.4

CAAATA…(aptamer)..TATCTT

2.5

OFF

ON

2.1

CTGATA..(aptamer)..TATGTA

1.5 2.2

ATGATA..(aptamer)..TATTTA

1.5

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SLIDE 21

21

gth treng

  • n S

nslatio Tran

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SLIDE 22

22

RBS Calculator

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SLIDE 23

23

th

Translation S h

Strengt

Strength

  • n

S

ΔG

nslatio

Particular

G

Tra

Particular Sequence

Ligand Concentration

Sequence

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SLIDE 24

Derivation of ΔG

A Thermodynamics based Model

24

A Thermodynamics‐based Model

: standby mRNA rRNA

G G   

mRNA

G 

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

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SLIDE 25

Derivation of ΔG

A Thermodynamics based Model

25

A Thermodynamics‐based Model

spacing

G 

: standby mRNA rRNA

G G   

start

G 

mRNA

G 

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

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SLIDE 26

The RBS Calculator

G

26

:

          

standby spacing tot m RNA m RNA rRNA start

G G G G G G

Translation Rate

tot

G

Ke 

 

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

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SLIDE 27

The RBS Calculator

Verification of Accuracy

27

Verification of Accuracy

Green Fluorescent Protein RBS AUG

Output Fluorescence Measurement p

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SLIDE 28

The RBS Calculator

Verification of Accuracy

28

Verification of Accuracy

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SLIDE 29

The RBS Calculator

→Forward Engineering

29

→Forward Engineering

Output Fluorescence Output Fluorescence (Translation Rate)

Green Fluorescent Protein RBS AUG

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SLIDE 30

The RBS Calculator →F d E i i

30

→Forward Engineering

rna1995, 51nt

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SLIDE 31

31

AND Gate Module

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SLIDE 32

Architecture of AND gate

32

Input1 Input2

Input 1

supD

T7

GFP

UAG

| |

RBS

T7ptag

* *

UAG UAG

Input 2

RBS

T7ptag

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SLIDE 33

Phase diagram

33

| |

RBS

T7 t

* * | |

RBS

T7ptag

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SLIDE 34

Step 1

34

TPP Hammerhead a e ead Ribozyme

* * | | RBS

T7ptag

* * RBS

T7ptag

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SLIDE 35

Result of step 1

35

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SLIDE 36

Step 2

36

p

| | * * | |

T7ptag

RBS

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SLIDE 37

Result of step 2

37

∆G= 1.33 kJ/mol ∆G= -0.4873 kJ/mol RBS: ACACAGGAC GGAGUGG RBS: ACACAGGAC GGAGUGG

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SLIDE 38

38

Bistable Switch Module Bistable Switch Module

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SLIDE 39

Construction of Bistable Switch

39

Construction of Bistable Switch

CI 434 mRFP CI eGFP mRFP eGFP

PRM PR

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SLIDE 40

Softcoding of Bistable Switch

40

CI 434 mRFP CI eGFP CI 434 mRFP eGFP

PRM PR

TPP hammerhead ribozyme TPP hammerhead ribozyme +TPP No TPP dG=4.6kJ/mol High TPP conc. dG=7.0kJ/mol

Translation strength of CI434

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SLIDE 41

Stochastic Model of Bistable Switch

41

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SLIDE 42

Stochastic Model of Bistable Switch

42

Fit with Softcoding Result

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SLIDE 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

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SLIDE 44

44

Fi i Fine‐tuning bi th ti th biosynthetic pathway

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SLIDE 45

Violacein pathway

45

deoxychromoviridans

N H N N H N H N H N H H

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SLIDE 46

Violacein pathway

46

deoxychromoviridans

N H N N H N H N H N H H

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SLIDE 47

Design

47

L‐tryptophan IPA imine

VioA VioB VioB

IPA imine dimer dimer P d i l i

VioE VioE

Prodeoxyviolacein

VioD VioD

Deoxy‐ chromoviridans Proviolacein chromoviridans

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SLIDE 48

Design

48

IPA imine dimer dimer

VioE

Prodeoxyviolacein

VioD

Deoxy‐ h i id Proviolacein chromoviridans

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SLIDE 49

Fine‐tuning pathway

49

Translation Strength control

violacein violacein violacein violacein

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SLIDE 50

Fine‐tuning pathway

50

Solvent ‐TPP TPP 10nM TPP 100nM TPP 1M TPP 10M

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SLIDE 51

Fine‐tuning pathway

51

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SLIDE 52

52

Summary

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SLIDE 53

Idea Summary

53

Idea Summary

Soft coding Soft-coding

Ligand Conc. Translation Strength RBS Sequence

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SLIDE 54

Achievements

54

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 t – AND gate – Bistable switch – Violacein bio‐synthesis

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SLIDE 55

Achievements

55

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 t – AND gate – Bistable switch – Violacein bio‐synthesis

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SLIDE 56

RNA Controllers

56

RNA Controllers

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SLIDE 57

Achievements

57

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 t – AND gate – Bistable switch – Violacein bio‐synthesis

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SLIDE 58

RBS Calculator

58

RBS Calculator

Green Fluorescent Protein RBS AUG Green Fluorescent Protein RBS AUG

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SLIDE 59

Achievements

59

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 t – AND gate – Bistable switch – Violacein bio‐synthesis

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SLIDE 60

Applications

60

Applications

CI 434 mRFP CI eGFP

PRM PR

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SLIDE 61

Prospect & future plans

61

Prospect & future plans

  • New generation

– Quantitative Q – Predictable Fast – Fast

  • Improvement

– Precise – General General

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SLIDE 62

Parts

62

  • Favorite Parts

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

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SLIDE 63

Human Practice

63

Human Practice

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SLIDE 64

Collaboration

64

Collaboration

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SLIDE 65

Lecture

65

Lecture

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SLIDE 66

Acknowledgement

66

Acknowledgement

  • Prof. Jorg Hartig
  • Prof. Ronald Breaker Prof. Yohei Yokobayashi
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SLIDE 67

Special thanks

67

Special thanks

  • Prof. Qi Ouyang
  • Prof. Ming Tian
  • Prof. Chongren Xu
  • Prof. Hongwei Guo

P f W h W i

  • Prof. Wensheng Wei
  • Prof. Xinxiang Zhang
  • Prof Luhua Lai
  • Prof. Luhua Lai
  • Prof. Lei Liu
  • Prof Peng Chen
  • Prof. Peng Chen
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SLIDE 68

68

THANK YOU FOR YOUR ATTENTION THANK YOU FOR YOUR ATTENTION