Regulatory Dynamicsin Engineered GeneNetworksThe Physicochemical Foundation Notes | EduRev

: Regulatory Dynamicsin Engineered GeneNetworksThe Physicochemical Foundation Notes | EduRev

 Page 1


Regulatory Dynamics
in Engineered Gene
Networks
The Physico-chemical Foundation
of Transcriptional Regulation with
Applications to Systems Biology
Mads Kærn
Boston University
Center for BioDynamics
Center for Advanced Biotechnology
Department of Biomedical Engineering
Page 2


Regulatory Dynamics
in Engineered Gene
Networks
The Physico-chemical Foundation
of Transcriptional Regulation with
Applications to Systems Biology
Mads Kærn
Boston University
Center for BioDynamics
Center for Advanced Biotechnology
Department of Biomedical Engineering
Disclaimer. This document contains material that has been reproduced from various sources
without permission from the copyright owners. As a result, the document may only be dis-
tributed to participants of the 4th International Systems Biology Conference, Washington Uni-
versity, St. Louis. All other material is c 
 Mads Kærn, 2003. The document is intended for
educational purposes only. Should any copyrights have been infringed, please contact the author
and the material will be removed immediately.
Page 3


Regulatory Dynamics
in Engineered Gene
Networks
The Physico-chemical Foundation
of Transcriptional Regulation with
Applications to Systems Biology
Mads Kærn
Boston University
Center for BioDynamics
Center for Advanced Biotechnology
Department of Biomedical Engineering
Disclaimer. This document contains material that has been reproduced from various sources
without permission from the copyright owners. As a result, the document may only be dis-
tributed to participants of the 4th International Systems Biology Conference, Washington Uni-
versity, St. Louis. All other material is c 
 Mads Kærn, 2003. The document is intended for
educational purposes only. Should any copyrights have been infringed, please contact the author
and the material will be removed immediately.
Contents
1 The Biology of Gene Expression 7
1.1 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2 Genes and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 Transcription and Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.1 Prokaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4 Regulation of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4.1 The Lactose Operon of E. coli . . . . . . . . . . . . . . . . . . . . . . 20
1.4.2 The Genetic Switch in Bacteriophage? . . . . . . . . . . . . . . . . . 22
1.4.3 The Galactose Regulon in S. cerevisiae . . . . . . . . . . . . . . . . . 24
2 Engineered Gene Networks 31
2.1 Some Tools of the Trade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.1 Cutting and Pasting DNA . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.2 Plasmid Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.1.3 Extracting DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Engineering Regulatory Modules . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.1 Genetic Switches in E. coli . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.2 Genetic Switches in S. cerevisiae . . . . . . . . . . . . . . . . . . . . 40
2.2.3 Mammalian Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3 Engineering Regulatory Circuits . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.4 How Transcriptional Regulation Works . . . . . . . . . . . . . . . . . . . . . 48
3 Modeling Small Gene Networks 51
3.1 Biochemical Reaction Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.1 Elementary Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.2 Law of Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.3 Generalized Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.1.4 Chemical Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1.5 The Michaelis-Menten Reaction . . . . . . . . . . . . . . . . . . . . . 60
3.1.6 Hill-type Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2 Modeling Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.3 Modeling cis-Regulatory Systems . . . . . . . . . . . . . . . . . . . . . . . . 69
3.3.1 Repressor-Operator Binding . . . . . . . . . . . . . . . . . . . . . . . 70
3.3.2 Alternative Reaction Paths . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.3 Cooperative Binding of Dimers . . . . . . . . . . . . . . . . . . . . . 74
Page 4


Regulatory Dynamics
in Engineered Gene
Networks
The Physico-chemical Foundation
of Transcriptional Regulation with
Applications to Systems Biology
Mads Kærn
Boston University
Center for BioDynamics
Center for Advanced Biotechnology
Department of Biomedical Engineering
Disclaimer. This document contains material that has been reproduced from various sources
without permission from the copyright owners. As a result, the document may only be dis-
tributed to participants of the 4th International Systems Biology Conference, Washington Uni-
versity, St. Louis. All other material is c 
 Mads Kærn, 2003. The document is intended for
educational purposes only. Should any copyrights have been infringed, please contact the author
and the material will be removed immediately.
Contents
1 The Biology of Gene Expression 7
1.1 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2 Genes and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 Transcription and Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.1 Prokaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4 Regulation of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4.1 The Lactose Operon of E. coli . . . . . . . . . . . . . . . . . . . . . . 20
1.4.2 The Genetic Switch in Bacteriophage? . . . . . . . . . . . . . . . . . 22
1.4.3 The Galactose Regulon in S. cerevisiae . . . . . . . . . . . . . . . . . 24
2 Engineered Gene Networks 31
2.1 Some Tools of the Trade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.1 Cutting and Pasting DNA . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.2 Plasmid Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.1.3 Extracting DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Engineering Regulatory Modules . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.1 Genetic Switches in E. coli . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.2 Genetic Switches in S. cerevisiae . . . . . . . . . . . . . . . . . . . . 40
2.2.3 Mammalian Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3 Engineering Regulatory Circuits . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.4 How Transcriptional Regulation Works . . . . . . . . . . . . . . . . . . . . . 48
3 Modeling Small Gene Networks 51
3.1 Biochemical Reaction Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.1 Elementary Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.2 Law of Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.3 Generalized Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.1.4 Chemical Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1.5 The Michaelis-Menten Reaction . . . . . . . . . . . . . . . . . . . . . 60
3.1.6 Hill-type Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2 Modeling Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.3 Modeling cis-Regulatory Systems . . . . . . . . . . . . . . . . . . . . . . . . 69
3.3.1 Repressor-Operator Binding . . . . . . . . . . . . . . . . . . . . . . . 70
3.3.2 Alternative Reaction Paths . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.3 Cooperative Binding of Dimers . . . . . . . . . . . . . . . . . . . . . 74
4
3.3.4 Synergism in RNA Polymerase Binding . . . . . . . . . . . . . . . . . 77
3.3.5 DNA looping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.4 Models of Gene Regulatory Systems . . . . . . . . . . . . . . . . . . . . . . . 83
3.4.1 The Lactose Operon in E. coli . . . . . . . . . . . . . . . . . . . . . . 83
3.4.2 The Galactose Regulon in S. cerevisiae . . . . . . . . . . . . . . . . . 89
3.5 Models of Engineered Gene Networks . . . . . . . . . . . . . . . . . . . . . . 93
3.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Page 5


Regulatory Dynamics
in Engineered Gene
Networks
The Physico-chemical Foundation
of Transcriptional Regulation with
Applications to Systems Biology
Mads Kærn
Boston University
Center for BioDynamics
Center for Advanced Biotechnology
Department of Biomedical Engineering
Disclaimer. This document contains material that has been reproduced from various sources
without permission from the copyright owners. As a result, the document may only be dis-
tributed to participants of the 4th International Systems Biology Conference, Washington Uni-
versity, St. Louis. All other material is c 
 Mads Kærn, 2003. The document is intended for
educational purposes only. Should any copyrights have been infringed, please contact the author
and the material will be removed immediately.
Contents
1 The Biology of Gene Expression 7
1.1 The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
1.2 Genes and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 Transcription and Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.3.1 Prokaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.3.2 Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.4 Regulation of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.4.1 The Lactose Operon of E. coli . . . . . . . . . . . . . . . . . . . . . . 20
1.4.2 The Genetic Switch in Bacteriophage? . . . . . . . . . . . . . . . . . 22
1.4.3 The Galactose Regulon in S. cerevisiae . . . . . . . . . . . . . . . . . 24
2 Engineered Gene Networks 31
2.1 Some Tools of the Trade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.1 Cutting and Pasting DNA . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.2 Plasmid Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.1.3 Extracting DNA Sequences . . . . . . . . . . . . . . . . . . . . . . . 36
2.2 Engineering Regulatory Modules . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.1 Genetic Switches in E. coli . . . . . . . . . . . . . . . . . . . . . . . . 39
2.2.2 Genetic Switches in S. cerevisiae . . . . . . . . . . . . . . . . . . . . 40
2.2.3 Mammalian Switches . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.3 Engineering Regulatory Circuits . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.4 How Transcriptional Regulation Works . . . . . . . . . . . . . . . . . . . . . 48
3 Modeling Small Gene Networks 51
3.1 Biochemical Reaction Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.1.1 Elementary Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.2 Law of Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.1.3 Generalized Mass Action . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.1.4 Chemical Equilibrium . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.1.5 The Michaelis-Menten Reaction . . . . . . . . . . . . . . . . . . . . . 60
3.1.6 Hill-type Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2 Modeling Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.3 Modeling cis-Regulatory Systems . . . . . . . . . . . . . . . . . . . . . . . . 69
3.3.1 Repressor-Operator Binding . . . . . . . . . . . . . . . . . . . . . . . 70
3.3.2 Alternative Reaction Paths . . . . . . . . . . . . . . . . . . . . . . . . 71
3.3.3 Cooperative Binding of Dimers . . . . . . . . . . . . . . . . . . . . . 74
4
3.3.4 Synergism in RNA Polymerase Binding . . . . . . . . . . . . . . . . . 77
3.3.5 DNA looping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
3.4 Models of Gene Regulatory Systems . . . . . . . . . . . . . . . . . . . . . . . 83
3.4.1 The Lactose Operon in E. coli . . . . . . . . . . . . . . . . . . . . . . 83
3.4.2 The Galactose Regulon in S. cerevisiae . . . . . . . . . . . . . . . . . 89
3.5 Models of Engineered Gene Networks . . . . . . . . . . . . . . . . . . . . . . 93
3.6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Foreword
The future success of System Biology requires the establishment of general principles
and the development of methodologies that can be used to link the behavior of individ-
ual molecules to system characteristics and functions. In order to achieve this goal, we
need to study systems that have been characterized in minute detail and are suf?ciently
small to be manageable. The central theme in this tutorial is the use of engineered gene
networks to deduce principles that govern gene transcription and to develop reasonably
accurate system level models from qualitative molecular level information. The tutorial
consists of three parts: (1) The Biology of Gene Expression, (2) Genetic Network Engi-
neering and (3) Modeling Small Gene Networks. No previous knowledge of molecular
biology is assumed.
The purpose of Part (1) is to provide a brief introduction to the fundamental biol-
ogy of gene expression and a discussion of the current theories of gene regulation in
bacteria and in yeast. Part (2) will provide a basic introduction to some experimental
techniques. The main emphasis, however, is a discussion of how genetically engineered
systems have provided support for the theories of transcriptional regulation introduced
in Part (1) and how they are used to investigate system level characteristics and function.
Part (3) discusses the physico-chemical basis of gene regulatory systems and provide
a detailed and rigorous methodology that can be used to convert qualitative molecular
level models into quantitative system level descriptions. Particular emphasis will be
given to the limits and dangers of quantitative modeling of which any researcher in
Systems Biology should be aware.
Discussions and comments from Michael Driscoll and Michael Thompson have
been very valuable during the writing of these notes. The reader is kindly reminded that
the notes serve only as a brief introduction to a very large subject area. They include
material that I believe is most relevance to readers that are involved in the mathematical
and computational aspects of Systems Biology, and are looking for a brief summary of
important aspects from molecular biology and physical chemistry. I have attempted to
present this material in a way that is accessible to a non-specialist audience. Despite
my best efforts, this has undoubtfully resulted in descriptions that in many aspects are
oversimpli?ed. My sincere apologies go to the authors of the books and the articles that
for one reason or the other did not make the Suggested Readings lists. Please report
errors and mistakes to mkaern@bu.edu. Suggestions that can be used to improve the
quality of future versions are most welcome.
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