Together with my colleagues, Harley McAdams and John Ross, I was deeply immersed in models of its networks to study the implications of molecular discreteness and noise in the robustness of cellular decisions. Chris took off on his own, deepening his understanding of Systems Biology through research on this model system. It is gratifying to see Lambda as an organizing example used so effectively in this text.
Even more than half a century after its discovery, there are still new mechanisms being discovered about the functioning of the Lambda switch. Thus, the classical biologists demand rightly so that anyone studying the switch must be an expert in biology and understand Lambda lore, in particular, quite well because otherwise any models of its architecture and function will not properly capture the mechanistic complexity or uncertainty about its operation.
The biochemists and biophysicists will insist that the particulars of the mechanisms—how exactly the proteins interact and degrade, how DNA loops to form a key component of the switch, how the complex thermodynamics of multicomponent promoter binding leads to the proper ordering of states for stabilizing the two decisions, how the stochastic effects due to the discrete nature of the chemistry leads to diversity in behavior—all must be considered carefully or formally discounted to justify explanations for how the system works.
The computational biologists and systems biologists require, on top of the above, deep understanding of biological data analysis, numerical algorithms, graph theory, and dynamical systems and control theory in order to build appropriate models justified by data and to analyze them properly, and a fine sense of how to interpret and abstract the principles of control buried in the dunes of detail about the system.
Students, throw up your hands! How do you start with all this deep and complex science and engineering to learn? As a first step, we must learn to live with our ignorance, and only then can we begin to fight to eradicate it. The frontier of science is where ignorance begins, and for Systems Biology, there is plenty of undiscovered land. This book by Professor Myers is one of the few texts in the area that gently brings the uninitiated to these edges. As such, biologists have had to draw assistance from those in mathematics, computer science, and engineering.
The result has been the development of the fields of bioinformatics and computational biology terms often used interchangeably. The major goal of these fields is to extract new biological insights from the large and noisy sets of data being generated by high-throughput technologies. Initially, the main problems in bioinformatics were how to create and maintain databases for massive amounts of DNA sequence data. Addressing these challenges also involved the development of efficient interfaces for researchers to access, submit, and revise data.
Bioinformatics has expanded into the development of software that also analyzes and interprets these data. While bioinformatics has come to mean many things, this text uses the term bioinformatics to refer to the analysis of static data such as DNA and protein sequences, techniques for finding genes or evolutionary patterns, and cluster analysis of microarray data.
Algorithms for bioinformatics are not covered in this text. The focus of this text is modeling, analysis, and design methods for systems biology. Systems biology has been the subject of several books Kitano, ; Alberghina and Westerhoff, ; Palsson, ; Wilkinson, ; Alon, ; Konopka, , each of which give it a somewhat different meaning.
This book uses the term to mean the study of the mechanisms underlying complex molecular processes as integrated into systems or pathways made up of many interacting genes and proteins. In other words, systems biology is concerned with the analysis of dynamic models. While it has long been known that developing dynamic models of complete systems is essential to understanding biological processes, it is only recently that the emergence of new high-throughput experimental data acquisition methods has made it possible to explore such computational models.
Some example experimental techniques include cDNA microarrays and oligonucleotide chips Brown and Botstein, ; Lipschutz et al. Systems biology involves the collection of large experimental data sets using high-throughput technologies, the development of mathematical models that predict important elements in this data, design of software to accurately and efficiently make predictions in silico i.
The ultimate goal of systems biology is to develop models and analytical methods that provide reasonable predictions of experimental results. While it will never replace experimental methods, the application of computational approaches to gain understanding of various biological processes has the promise of helping experimentalists work more efficiently. These methods also may help gain insight into biological mechanisms, information which may not be obtained from any known experimental methods. Eventually, it may be possible that such models and analytical techniques could have substantial impact on our society such as aiding in drug discovery.
Systems biologists analyze several types of molecular systems, including genetic regulatory networks, metabolic networks, and protein networks. The primary focus of this book is genetic regulatory networks referred to as genetic circuits in this book. These circuits regulate gene expression at many molecular levels through numerous feedback mechanisms. Chapter 1 presents the basic molecular biology and biochemistry principles that are needed to understand these circuits. A few bacterial genetic circuits are well understood.
This circuit is described in Chapter 1 and used as a running example throughout this book. During the genomic age, standards for representing sequence data were and still are essential. Data collected from a variety of sources could not be easily used by multiple researchers without a standard data format. For systems biology, standard data formats are also being developed.
One format that seems to be gaining some traction is the systems biology markup language SBML see http: All the types of models described in this book can be reduced to a set of bio-chemical reactions. The basic structure of an SBML model is a list of chemical species coupled with a list of chemical reactions. Each chemical reaction includes a list of reactants, products, and modifiers. It also includes a mathematical description of the kinetic rate law governing the dynamics of this reaction. SBML is not a language for use in constructing models by hand.
Fortunately, several graphical user interfaces GUIs have been developed for entering or drawing up chemical reaction networks, which then can be exported in the SBML format. Databases have been developed to store different sets of information ranging from nucleotide sequences within GenBank http: Recently, there has also been developed a new database for SBML models of various biochemical systems http: A final essential piece of the puzzle is the development of tools for analysis. This text concentrates on describing the methods used by such tools.
Given our vast experience in reasoning about complex circuits and systems, engineers are uniquely equipped to assist with the development of tools for the modeling and analysis of genetic circuits. It has been shown that viewing a genetic circuit as an electronic circuit can yield new insights and open up the application of engineering tools to genetic circuits McAdams and Shapiro, Therefore, as in the sequencing of the human genome, collaborations between engineers and biologists will be essential to the success of systems biology.
The major goal of this textbook is to facilitate these collaborations. An engineering approach involves three parts as shown in Figure 0. First, engineers examine experimental data in order learn mathematical models. Second, engineers develop efficient abstraction and simulation methods to analyze these models. Finally, engineers use these analytical methods to guide the design of new circuits. This book discusses all three aspects of this engineering approach as applied to genetic circuits.
Chapter 2 describes modern experimental techniques and methods for learning genetic circuit models from the data generated by these experiments. The next four chapters explore methods for analyzing these genetic circuit models. Perhaps, the most common method for modeling and analysis uses differential equations, a topic which is the subject of Chapter 3. In genetic circuits, however, the numbers of molecules involved are typically very small, thereby requiring the use of stochastic analysis; these methods are described in Chapter 4. Stochastic analysis can be extremely complex limiting its application often to only the simplest systems.
To reduce this complexity, Chapter 5 presents several reaction-based abstraction methods to simplify the models while maintaining reasonable accuracy. Since the state space of these models is still often quite large, Chapter 6 presents logical abstraction to reduce this state space and further improve analysis time.
Finally, using these analytical methods, researchers are beginning to design synthetic genetic circuits as described in Chapter 7. It is our hope that this book will prove to be useful to both engineers who wish to learn about genetic circuits and to biologists who would like to learn about engineering techniques that can be used to study their systems of interest. Engineering approach to modeling, analysis, and design of genetic circuits. In a semester version of this course, students select their own biological circuit and learn about them from research papers.
Students then repeat the model design or analysis using their selected circuit. Throughout, the students use our iBioSim tool, which supports most of the topics discussed in this textbook. This tool allows one to construct models, learn from experimental data, and perform either differential or stochastic analyses utilizing automatic reactionbased and logical abstractions. Course examples including lecture materials and assignments as well as our iBioSim software are freely available from: Michael Samoilov of the University of California at Berkeley.
When Michael and I were graduate students at Stanford University, he introduced me to Adam, thus launching many discussions about the relationship between asynchronous electronic circuits and genetic circuits. The result was Adam joining my dissertation committee. In , during my sabbatical at Stanford University, we revisited these conversations, thus triggering much of the research described in this textbook.
I would also like to thank Professor David Dill of Stanford who hosted my sabbatical and used a draft of this text in one of his courses. Finally, I would like to thank Daniel Gillespie for several stimulating discussions. Much of the work described in this textbook was conducted by my graduate students. Nathan Barker, now an Assistant Professor at Southern Utah University, developed the causal model learning methods described in Chapter 2. Nam Nguyen, now a Ph. All three of these graduate students as well as my current students, Curtis Madsen and Kevin Jones, have been involved in the development of iBioSim, the tool that implements many of the methods described in this textbook.
I would also like to thank Baltazar Aguda of Ohio State University, Lingchong You of Duke University, and the other anonymous reviewers whose comments helped improve this book. Finally, I would especially like to thank my family, Ching and John, for being patient with me during the writing process. Without their love and support, this book would not have been possible. Obviously, the material covered in this chapter is quite basic as it is usually the topic of whole courses. It should, however, give the grounding necessary to begin studying the modeling, analysis, and design of genetic circuits.
This chapter begins with a discussion of chemical reactions, the basic mechanism used by all life processes. Although there are more than a different types of atoms, about 98 percent of the mass of any living organism is made up of only six types: It is truly amazing what nature has constructed from such a simple set of basic building blocks. All materials that make up a living organism are created or destroyed via chemical reactions. Chemical reactions combine atoms to form molecules and combine simpler molecules to form more complex ones. Atoms form molecules 1 2 Engineering Genetic Circuits by binding together via covalent, ionic, and hydrogen bonds.
Chemical reactions can also work in reverse to turn complex molecules into simpler molecules or atoms. A simple chemical reaction for the formation of water from hydrogen and oxygen is shown below: In this equation, the subscripts in H2 and O2 indicate that the hydrogen and oxygen are present in dimer form i. The molecules H2 and O2 are known as the reactants for this reaction. The water molecule, H2 O, is composed of two hydrogen atoms and one oxygen atom, and it is known as the product for this reaction.
The number 2 in front of H2 and H2 O indicates that two hydrogen dimers are used in the reaction and two water molecules are produced. These numbers along with the implicit one in front of O2 are known as the stoichiometry of the reaction. Since matter must be conserved by chemical reactions, the numbers of each atom on both sides of the equation must be equal. Note that many chemical reactions shown in this book may not have this property when reactants or products are not listed to simplify the presentation.
The k above the arrow is known as the rate constant for this chemical reaction.
Editorial Reviews
This value indicates how likely or how fast this reaction typically occurs. The rate constant is used in many of the modeling techniques described in this book, but unfortunately, it is often difficult to determine accurately for bio-chemical reactions. The rate of a chemical reaction is governed by the law of mass action. Namely, the rate of a reaction is determined by this rate constant and the concentrations of the reactants raised to the power of their stoichiometry. Using the law of mass action for the reaction above, the rate of water formation via this reaction is: The 2 in front of the k signifies that each reaction produces two molecules of water.
Chemical reactions must obey the laws of thermodynamics. The first law states that energy can be neither created nor destroyed, and the second law states that entropy i. These two laws can be combined into a single equation: Willard Gibbs who introduced this concept in Consider a reversible reaction of the form: When this value is negative, the forward reaction can occur spontaneously. When this value is positive, the reverse reaction can occur spontaneously. When this value is zero, the reaction is in a steady state i. From the above discussion, it would appear that chemical reactions with a positive free energy cannot occur.
However, free energies of chemical reactions are additive. In other words, a reaction with a positive free energy can occur if there exists another reaction or reactions in the system that when their free energies are added together result in a cumulative negative free energy. One of the most common reactions that is used for this purpose is the hydrolysis of adenosine triphosphate ATP given by the equation below: The forward reaction releases energy i. Coupling the forward reaction above with other reactions that have a positive free energy can allow these reactions to occur.
These types of ATP reactions occur in all living organisms. Therefore, ATP is known as the universal energy currency of living organisms. Even reactions with a negative free energy may not occur spontaneously as there is typically an energy barrier known as the activation energy that must be overcome first. For example, a collision between two molecules is 4 Engineering Genetic Circuits typically necessary to get them close enough to break existing chemical bonds.
The amount of these collisions can be increased by applying heat. Another mechanism is the use of an enzyme. An enzyme, or catalyst, is a reactant that accelerates a reaction without being consumed by the reaction. This text also uses the term modifier for such a chemical species to differentiate it from reactants which are consumed by reactions.
It is typically the case that the amount of enzyme is much smaller than that of the other reactants, also known as substrates. Note that enzymes do not effect the overall free energy of the reaction, but rather they simply help the reaction overcome its activation energy barrier. There are four kinds of macromolecules: Carbohydrates are made up of carbon and water, so their chemical formula is basically Cn H2 O m , where m and n are often equal or nearly so.
Carbohydrates are also called sugars. An example carbohydrate is glucose. Carbohydrates are an important source of chemical energy that is used to power biological processes. The energy stored in their bonds is used to power everything from cell movement to cell division to protein synthesis. Lipids are made up of mostly carbon and hydrogen atoms.
They often have both a hydrophilic water-loving part and a hydrophobic water-fearing part. Their primary use is to form membranes. Membranes separate cells from one another and create compartments within cells as well as having other functions. Lipids make good membranes because their hydrophobic parts attract to form lipid bi-layers where the exterior allows water, but their interior repels water. This property allows the lipid bi-layers to form between areas containing water, but they do not allow water to easily pass through.
Examples of simple lipids include fats, oils, and waxes. Nucleic acids are the macromolecules that store information within living organisms. A nucleic acid, or nucleotide, is composed of a chemical base bound to a sugar molecule and a phosphate molecule. A sequence of nucleic acids connected by their phosphate molecules encodes the instructions to construct the product produced by a gene. Nucleic acids can be composed together in one of two forms: A strand of DNA is composed of four types of bases: Adenine and guanine are known as purines while cytosine and thymine are known as pyrimidines.
These bases are each composed of oxygen, carbon, nitrogen, and hydrogen as shown in Figure 1. A strand of DNA is always read in one direction. DNA is a double-stranded molecule as it consists of two strands running in opposite directions. The base pairs A-T and G-C are complementary in that if an A is found on one strand, then a T is found on the other strand in the same location. This feature is due to the fact that A bound to T and G bound to C are so strongly thermodynamically favored that any other combination is highly unlikely.
This base pairing property allows one strand of DNA to serve as a template for synthesizing another complementary strand which greatly facilitates making copies of DNA sequences. The chemical makeup of this base pairing creates a force that twists the DNA into its well-known coiled double helix structure. RNA has many uses. This mRNA molecule is used to carry the genetic information encoded in the DNA to the ribosome, the protein assembly machinery.
The mRNA molecule is then used by the ribosome as a template to synthesize the protein that is encoded by the gene. Proteins are the basic building blocks of nearly all the molecular machinery of an organism. A cell may contain thousands of different proteins. Protein molecules are made from long chains of amino acids. There are 20 different kinds of amino acids found in living organisms. Genes in the DNA specify the order of the amino acids that make up the protein. During transcription, the code for a protein is transferred by using the DNA as a template to construct a strand of mRNA.
This mRNA then proceeds to a ribosome where it is translated into a protein. A ribosome constructs a protein one amino acid at a time in the order specified by the codons in the mRNA. A codon is a group of three bases which specifies a particular amino acid using the genetic code shown in Table 1. The first codon UUU was associated with the amino acid phenylalanine by Nirenberg and Matthaei in Notice in the table that most amino acids are associated with more than one codon.
These redundant codons often differ in the third position as is the case for the codons for Valine i. National Human Genome Research Institute. Instead, they are used to tell the ribosome when the protein is complete and translation should stop. After a protein is constructed, it folds into a specific three-dimensional configuration. The shape and position of the amino acids in this folded state determines the function of the protein. Therefore, understanding and predicting protein folding has become an important area of research.
The structure of a protein is described in four levels as shown in Figure 1. The primary structure is simply the sequence of amino acids that make up the protein. The secondary structure is the patterns formed by the amino acids that are near to each other. The ternary structure is the arrangement of amino acids that are far apart. Finally, the quaternary structure describes the arrangement of proteins that are composed of multiple amino acid chains.
Alpha helix Quaternary protein structure is a protein consisting of more than one amino acid chain. Namely, information encoded in the DNA within its genome is used to produce RNA which produces the proteins that all organisms need. This amazing fact gives substantial support to the notion that all life began from a common origin. A genome is divided into genes where each gene encodes the information necessary for constructing a protein or possibly an RNA molecule using the genetic code shown in Table 1.
Some of these proteins also control the timing for when other genes should produce their proteins as described later in Section 1. The term gene, which was introduced in by the Danish botanist Wilhelm Johanssen, means the hereditary unit found on a chromosome where a chromosome is a linear DNA molecule. Genes, however, were discovered 50 years earlier by Gregor Mendel, though he called them factors. He was an Austrian monk who experimented with his pea plants in the monastery gardens. Since pea plants have both male and female organs, they normally self-fertilize. However, with the use of a pair of clippers, he was able to control the plants parents and ultimately their traits.
It was a remarkable discovery that went largely ignored for nearly 50 years until the late 19th century when three researchers essentially duplicated his results. Initially, it was not completely accepted that genes are part of DNA. This discovery showed that DNA is composed of two strands composed of complementary bases. This base pairing idea shed light on how DNA could encode genetic information and be readily duplicated during cell division.
Between and , work by Crick and others showed how DNA codes for amino acids and thus proteins. At the turn of this century, another major milestone was accomplished. In February , two largely independent drafts of the human genome were published International Human Genome Sequencing Consortium, ; Venter et al. Both drafts predicted that the human genome contains 30, to 40, genes though now believed to be about 20, to 25, They made this estimate by using computer programs to analyze their sequence database to count open reading frames, i. Estimates have been improved in recent years using partial mRNA sequences to precisely locate genes by aligning the start and end portions with sequences in a DNA sequence database.
The DNA also contains regulatory sequences that mark the start and end of genes and those used to switch genes on and off, but they also account for only a very small amount of the DNA. About 40 to 45 percent of our genome is composed of repetitive DNA, short sequences often repeated s of times.
Although they have no known role in the synthesis of proteins, they are an excellent marker for identifying people. Junk DNA may have once contained real genes that are no longer functional due to mutations, and may form a record of our evolutionary history. In the human genome as well as that of all multicellular organisms, genes are not even continuous sequences.
The exons, or coding portions of a gene, are broken up as shown in Figure 1. Although both exons and introns are transcribed into mRNA, the introns are snipped out before the mRNA is translated into a protein. The purpose of introns remains unclear, but they may serve as sites for recombination i.
To complicate matters further, about 40 percent of genes are alternatively spliced, meaning that the part that is considered introns may vary. The result is that a single gene can actually code for multiple proteins. One possible reason for alternative splicing is that it may reduce the chances of harmful mutations being passed to the next generation. If there are less genes, then there is a lower probability that a mutation affects an actual gene.
If it does affect a gene, it likely affects the correct production of several proteins derived from alternative splices, thereby reducing the likelihood that the individual survives to produce a new generation. Extensive use of alternative splicing may also explain how humans with 20, to 25, genes are so much more complicated than the roundworm and fruit fly that have comparable genome sizes. On the one hand, some organisms are composed of only a single cell.
Bacteria are one such type of unicellular organism. On the other hand, multicellular organisms are composed of many cells. In fact, humans are estimated to be made up of more than ,,,, cells! Each cell must be capable of taking in nutrients, converting these into energy, eliminating waste products, growing and developing, responding to various stimuli from their environment, and forming a complete copy of itself i.
Within each cell, the instructions for performing these tasks are contained in its genome. Introns and exons courtesy: Life on earth began about 3. Prokaryotes are unicellular organisms that lack a nuclear membrane and have few, if any, organelles. Most of the functions performed by organelles in higher organisms are performed by the prokaryotic cell plasma membrane. The major features of prokaryotic cells are shown in Figure 1. These features include external appendages such as the whip-like flagella for locomotion and the hair-like pili fimbriae for adhesion. The cell envelope consists of a capsule, a cell wall, and a cell plasma membrane.
The DNA of the prokaryotic chromosome is organized in a simple circular structure. Prokaryotes do not develop or differentiate into multicellular organisms. Bacteria are the best known and most studied form of prokaryote. While some bacteria do grow in masses, each cell exists independently. Bacteria can inhabit almost any place on earth. Major features of a a prokaryote and b a eukaryote courtesy: Eukaryotes appear in the fossil record about 1. Eukaryotes include fungi, mammals, birds, fish, invertebrates, plants, and complex single-celled organisms.
Eukaryotic cells are also about 10 times larger than prokaryotes, and hence their volume is up to times larger. While eukaryotes use the same genetic code and metabolic processes as prokaryotes, they have a higher level of organizational complexity which allows for the development of multicellular organisms. Eukaryotes also include many membrane-bounded compartments, known as organelles, where metabolic activities occur.
The major features of eukaryotic cells are shown in Figure 1. First, there is the cell plasma membrane, which provides a barrier between a eukaryotic cell and its environment. This membrane is a complex composed of lipids, proteins, and carbohydrates. Next, there is the cytoskeleton which is composed of microfilaments long thin fibers and microtubules hollow cylinders. The cytoskeleton gives a cell its shape and holds organelles in place.
It also helps during endocytosis i. Lastly, there is the cytoplasm which is the large fluid-filled space inside the cell. While in prokaryotes, the cytoplasm does not have many compartments, in eukaryotes, it includes many organelles. The cytoplasm is essential to a cell as it serves many functions including dissolving nutrients, breaking down waste products, and moving materials around the cell. For example, the nucleus of a human 12 Engineering Genetic Circuits cell has 23 chromosomes. In eukaryotes, the nucleus is the location where DNA replication and transcription takes place where as in prokaryotes, these functions occur in the cytoplasm.
The nucleus has a spheroid shape and is enclosed by a double membrane called the nuclear membrane, which isolates and protects the genome from damage or interference from other molecules. During transcription described in Section 1. Another important organelle is the ribosome, which is a protein and RNA complex used by both prokaryotes and eukaryotes to produce proteins from mRNA sequences.
A ribosome is a large complex composed of structural RNA and about 80 different proteins. During translation also described in Section 1. Due to the importance of protein synthesis, there are s or even s of ribosomes in a single cell. Ribosomes are found either floating freely in the cytoplasm or bound to the endoplasmic reticulum ER.
The ER is a network of interconnected membranes that form channels within a cell. It is used to transport molecules that are either targeted for modifications or for specific destinations. There is a rough ER and a smooth ER. The rough ER is given its rough appearance by the ribosomes adhered to its surface. The mRNA for proteins that either stay in the ER or are exported from the cell are translated at these ribosomes.
The smooth ER receives the proteins synthesized at the rough ER. Proteins to be exported from the cell are passed to the Golgi apparatus to be processed further, packaged, and transported to other locations. Mitochondria and chloroplasts are organelles that generate energy. Mitochondria are self-replicating and appear in various shapes, sizes, and quantities.
They have both an outer membrane that surrounds the organelle and an inner membrane with inward folds known as cristae that increase its surface area. Chloroplasts are similar, but they are only found in plants. They also have a double membrane and are involved in energy metabolism. Mitochondria and chloroplasts have their own genome which is a circular DNA molecule. The mitochondrial genome is inherited from only the mother.
It is believed that this DNA may have come from bacteria that lived within the cells of other organisms in a symbiotic fashion until it evolved to become incorporated within the cell. Although the mitochondrial genome is small, its genes code for some important proteins. For example, the mitochondrial theory of aging suggests that mutations in mitochondria may drive the aging process.
Lysosomes and peroxisomes are responsible for degrading waste products and food within a cell. They are spherical, bound by a membrane, and contain digestive enzymes, proteins that speed up biochemical processes. Since these enzymes are so destructive, they must be contained in a membrane-bound compartment. Peroxisomes and lysosomes are similar, but peroxisomes can replicate themselves while lysosomes are made in the Golgi apparatus.
Humans have between 20, and 25, genes while mustard grass has 25, known genes. Increased complexity can be achieved by the regulatory network that turns genes on and off. This network precisely controls the amount of production of a gene product, and it can also modify the product after it is made. Genes include not only coding sequences that specify the order of the amino acids in a protein, but also regulatory sequences that control the rate that a gene is transcribed.
These regulatory sequences can bind to other proteins which in turn either activate i. Transcription is also regulated through post-transcriptional modifications, DNA folding, and other feedback mechanisms.
Transcriptional regulation allows mRNA to be produced only when the product is needed. This behavior is quite analogous to electrical circuits in which multiple input signals are processed to determine multiple output signals. Thus, in this text, these regulatory networks are known as genetic circuits. Eukaryotic cells have three different RNA polymerases.
Transcription is initiated when a subunit of RNAP recognizes and binds to the promoter sequence found at the beginning of a gene. A promoter sequence is a unidirectional sequence that is found on one strand of the DNA. There are two promoter sequences upstream of each gene, and the location and base sequence of each promoter site varies for prokaryotes and eukaryotes, but both are recognized by RNAP. After binding to the promoter sequence, RNAP unwinds the double helix at that point and begins to synthesize a strand of mRNA in a unidirectional manner.
This strand is complementary to one of the strands of the DNA, and so it is known as the antisense or template strand. The other strand is known as the sense or coding strand. The transcription process terminates when the RNAP reaches a stop signal. Termination of transcription in eukaryotes is not fully understood.
The ability of RNAP to bind to the promoter site can be either enhanced or precluded by other proteins known as transcription factors. These proteins recognize portions of the DNA sequence near the promoter region known as operator sites. An overview of transcription and translation courtesy: In other words, an activator turns on or enhances gene expression while a repressor turns off or reduces gene expression. The effects of transcription factors can affect both adjacent genes known as cis-acting or distant genes known as trans-acting. Transcription can also be regulated by variations in the DNA structure and by chemical changes in the bases where the transcription factors bind.
For example, methylation is a chemical modification of the DNA in which a methyl group -CH3 is added. Methylation often occurs near promoter sites where there is cytosine preceded by guanine bases. The next step is protein synthesis from the mRNA by the translation process shown in Figure 1.
Translation is performed by the ribosomes using tRNA. Each tRNA has an anti-codon site that binds to a particular sequence of three nucleotides known as a codon. A tRNA also has an acceptor site that binds to the specific amino acid for the codon that is associated with the tRNA. Ribosomes are made up of a large subunit and a small subunit. Translation is initiated when a strand of mRNA meets the small subunit. The large subunit has two sites to which tRNAs can bind.
The A site binds to a new tRNA which comes bound to an amino acid. The translation process from mRNA into a polypeptide chain involves three steps: Next, a tRNA bound to methionine binds to this start signal beginning the elongation process. This process continues until translation comes to one of the three stop codons, that signals that translation should move into the termination step.
There is no tRNA that binds to the stop codon which signals the ribosome to split into its two subunits and release the newly formed protein and the mRNA template. At this point, the protein may undergo post-translational modifications while the mRNA is free to be translated again. A single mRNA transcript may code for many copies of a protein before it is degraded. It may even be transcribed by multiple ribosomes at the same time.
Translation can be regulated by the binding of repressor proteins to the mRNA molecule. Translational regulation is heavily utilized during embryonic development and cell differentiation. Types of viruses courtesy: National Center for Biotechnology Information. Their genomes include genes to produce their protein package and those required for reproduction during infection. Since viruses must utilize the machinery and metabolism of a host cell to reproduce, they are known as obligate intracellular parasites. Before entering the host, the virus is known as a virion, or package of genetic material.
A virion can enter a host through direct contact with an infected host or by a vector, or carrier. There are several types of viruses such as those shown in Figure 1. Bacteriophages are those that infect bacteria while animal viruses and retroviruses infect animals and humans. The main goal of a virus is to replicate its genetic material. There are five main stages to virus replication: During penetration, bacteriophages make a small hole in the cell wall and inject their genome into the cell leaving the virus capsid outside.
Animal viruses and retroviruses, such as HIV, enter their host via endocytosis. During the replication stage, the virus begins the destructive process of taking over the cell and forcing it to produce new viruses. Retroviruses synthesize a complementary strand of DNA using the enzyme reverse transcriptase, which can then be replicated using the host cell machinery.
During this stage, the virus also instructs the host to construct a variety of proteins which are necessary for the virus to reproduce. First, early proteins are produced which are enzymes needed for nucleic acid replication. Next, late proteins are produced that are used to construct the virus capsid.
Finally, lytic proteins are produced, if necessary, to open the cell wall for exit. During the assembly stage, the virus parts are assembled into new viruses simply by chance or perhaps assisted by additional proteins known as molecular chaperons. During the release stage, assembled viruses leave the cell either by exocytosis or by lysis.
Bacteriophages, on the other hand, typically must lyse, or break open, the cell to exit. These viruses have a gene that codes for the enzyme lysozyme that breaks down the cell wall causing it to swell and burst killing the host cell. The new viruses are then released into the environment. He also found that some of the newly infected E. It is a bacteriophage that infects E.
The lysis strategy uses the machinery of the E. It then lyses the cell wall killing the cell and allowing the newly formed viruses to escape and infect other cells. The lysogeny strategy is a bit more subtle. Its DNA is then replicated through the normal process of cell division. If after perhaps many generations it detects the eminent demise of its host, it can revert to the lysis strategy to produce new viruses that escape to infect other cells.
Over the years, much has been learned about this simple genetic circuit. It serves to both illustrate the concepts involved in such genetic circuits as well as a running example to explain the analysis methods presented in later chapters. CI monomers are produced from the cI gene.
These operator sites are overlapped by two promoters. In this case, RNAP bound to this promoter transcribes to the right producing transcripts from the cro gene. These two promoters form a genetic switch since transcripts can typically only be produced in one direction at a time. A single molecule, or monomer, of CI is composed of a carboxyl C and amino N domain connected by a chain of 40 amino acids. Two CI monomers react to form a dimer, CI2. This process is shown in Figure 1. Similarly, the cro gene codes for the Cro protein which also dimerizes in order to bind to OR operator sites as shown in Figure 1.
Cro monomers are produced from the cro gene.
Buy for others
Two Cro monomers form a Cro dimer which can bind to one of the OR operator sites. CI2 also serves as an activator when it is bound to OR 2. Although shown in black-and-white terms, things in biology are rarely so clear-cut. The rate of transcription in the scenario shown in Figure 1. It has no effect, however, on PR. While the PRM promoter producing transcripts for the CI molecule is initially inactive, the PR promoter producing transcripts for the Cro molecule is initially active as shown in Figure 1.
Its promoter has a stronger affinity for RNAP. Finally, if Cro2 happens to also bind to either operator sites OR 1 or OR 2, it represses its own production see Figure 1. While CI2 and Cro2 can bind to any of the three operator sites at any time, they have a different affinity to each site. The Cro2 has the reverse affinity as shown in Figure 1. Namely, it is likely to first bind to OR 3 to turn off CI production. Finally, in very high concentration, Cro2 can be found bound to all three sites. The effect of CI2 and Cro2 bound to each operator site.
Likely position of Cro2 bound to OR versus Cro2 concentration. At moderate concentration, it is equally likely to be also bound to b OR 2 or c OR 1. Cooperativity of CI2 binding. As mentioned above, the first dimer to bind to OR typically binds to OR 1. Next, this dimer helps attract another CI2 molecule onto the OR 2 site.
It does this by bending over such that one carboxyl domain from each dimer touch as shown in Figure 1. This effect is known as cooperativity, and it is so strong that it appears almost as if the two dimers bind simultaneously. The two dimers bound in this way have a dual effect as shown in Figure 1. Namely, they repress production of Cro and they activate production of CI. While not often found in wild-type i. The effect of cooperativity is that the repression of Cro by CI becomes more switch like.
The one without cooperativity is also controlled by CI monomers rather than dimers in that dimerization is also a form of cooperativity. The cooperative switch is very stable to small perturbations around this value. Effect of cooperativity of CI2 molecules on repression of Cro production. However, when the concentration of CI drops significantly, the repression of Cro production is rapidly released. In the non-cooperative switch, repression drops off much more gradually, making the transition to lysis much less sharp. Given the above discussion, at low to moderate concentrations of CI and Cro, there are three common configurations.
First, there may be no molecules bound to OR. In this case, Cro is produced at its full rate of production while CI is only produced at its low basal rate. In this case, Cro production is repressed and CI production is activated. Third, a Cro2 molecule may be bound to OR 3. In this case, CI cannot be produced even at its basal rate while Cro continues to be produced.
Therefore, the feedback through the binding of the products as transcription factors coupled with the affinities described makes the OR operator behave as a genetic bistable switch. In one state, Cro is produced locking out production of CI. In this state, the cell follows the lysis pathway since genes downstream of Cro produce the proteins necessary to construct new viruses and lyse the cell.
In the other state, CI is produced locking out production of Cro. In this state, the cell follows the lysogeny pathway since proteins necessary to produce new viruses are not produced. In the lysogeny state, the cell develops an immunity to further infection. The cleaved CI monomers are unable to dimerize and bind to OR 1 which reduces the concentration of CI2 molecules allowing Cro production to begin.
Once a cell commits to lysogeny, it becomes very stable and does not easily change over to the lysis pathway.
- !
- Audens World.
- Lord Bangboomboo.
An induction event is necessary to cause the transition from lysogeny to lysis. For example, as described earlier, lysogens i. UV light creates DNA damage that is potentially fatal to the cell.
Engineering Genetic Circuits
It also has the effect of cleaving the CI monomer into two parts as shown in Figure 1. This inactivates the CI molecule making it incapable of forming a dimer and binding to OR. As the concentration of complete CI molecules diminish, the cell is unable to maintain the lysogeny state. Cro production begins again moving the cell into the lysis pathway.
How do these proteins locate these sequences from amongst the millions within the bacteria? The second column of Table 1. The top line of each row is one strand of the DNA while the bottom line is the complementary strand. The base pair shown in lower case represents the midpoint of the sequence. Observing from this midpoint, one finds that a strand on one side of the midpoint is nearly symmetric with the complementary strand on the other side.
This may not, however, be readily obvious. The last row accumulates frequencies of each base pair in each position. The most likely entries form the following consensus sequence: For example, there is always an A in the 2nd and 16th position, a C in the 4th and 14th position, and nearly always a C in the 6th and 12th position. There are, however, some differences.
It is these differences that cause the differences in affinity for CI2 and Cro2 for the different operators. Notice that the first half of the operator sites OR 1 and OR 3 agree perfectly with the consensus sequence. The second half, however, has several differences which lead to different affinities for CI2 and Cro2. To see how CI2 and Cro2 recognize these operator sites differently, it is necessary to consider their structures in more detail.
These figures also show the attractions between these amino acids and the bases within the second half of the sequences for OR 1 and OR 3 note that they are reversed and inverted from how they are shown in Table 1. Both CI2 and Cro2 begin with the amino acid glutamine gln which is attracted to the A-T base pair in the second position. They also agree in the second amino acid serine ser which is drawn to the C-G base pair in the fourth position.
This commonality shows why CI2 and Cro2 are both attracted to these operator sites. They differ, however, in the remaining amino acids. The case is the reverse for OR 1. Note that as shown in Figure 1. RNAP like transcription factors must locate a particular sequence associated with a promoter on which to bind. There are many promoters each associated with one or more genes on a strand of DNA. Considering the sequences associated with these promoters, one can generate a consensus sequence indicating the most likely i.
It turns out that the most important part of the sequence are the 6 base pairs located near the and positions where means 35 base pairs away from the start of the gene. Comparing these portions of the consensus sequence with the same portions of the PRM promoter and the PR promoter, it is found that the PR promoter is a better match to the consensus sequence than PRM differing in only two bases while PRM differs in four. The first step is circularization. At each end of this strand is 12 bases of single stranded DNA known as cohesive, or sticky, ends which join to form a circular strand of DNA.
The location marked cos is the location of the cohesive ends. Continuing in a clockwise direction, the next 22 genes encode proteins that construct the head and tail of the virus. The site labeled attp, or attachment site, is where the DNA is split when it in integrated within the E. Next, comes five individual genes labeled, cIII, N, cI, cro, and cII which are used to make the decision between lysis and lysogeny as described below.
The Q gene is used during the lysis pathway. Finally, the three genes labeled lysis are used to lyse, or open, the bacteria. Transcripts from PRM are always terminated immediately after transcribing the cI gene. Transcripts from PR , however, encounter a terminator switch after transcribing the cro gene and can continue to transcribe cII, O, P, and Q genes.
This potential transcription is indicated with a dashed line. The promoter PL begins transcription for the N gene. Transcripts beginning at PL also encounter a terminator switch and can potentially continue to transcribe cIII, xis, and int genes. The int gene can also be transcribed starting from the PI promoter.
The PR0 promoter transcribes the genes for the proteins needed for the lysis pathway. The Pantiq promoter produces reverse transcripts for the gene Q.
Engineering Genetic Circuits - CRC Press Book
Finally, the PRE promoter can produce transcripts of the cI gene as well as reverse transcripts for the cro gene. The remainder of this section describes this complete circuit in more detail. The N and cro genes are the only ones that are active very early. The other genes such as cI do produce transcripts, but only at a low basal rate initially. Recall that a buildup of Cro can trigger the lysis pathway to be taken. The protein N is known as an anti-terminator, and its role is illustrated in Figure 1. RNAP that binds to the promoter PL known as the left promoter since transcripts move from the left of the gene cI transcribes the gene N, and it then hits a terminator switch as shown in Figure 1.
This action blocks transcription about 80 percent of the time such that all genes downstream of the switch do not get transcribed, and the proteins that they code for do not get produced. The result is that the RNAP now passes over the terminator switch and continues to transcribe the remaining genes as shown in Figure 1.
As mentioned above, there is another terminator switch that blocks transcripts to the right, and it is also controlled by the N protein. The transition from the very early to the early stage is marked by a buildup of the protein N. As just described, the protein N closes the terminator switches allowing transcripts from the PR and PL promoters to transcribe additional genes see the dashed arrows in Figure 1. Transcripts beginning from the PL promoter now proceed past its terminator switch to transcribe the cIII, xis, and int genes.
These transcripts also continue on to transcribe an important non-genetic portion of the DNA known as sib. The sib portion of the mRNA forms a hairpin as shown in Figure 1. Since the Int portion is destroyed before the Xis portion, more of the Xis protein is allowed to be synthesized than Int as shown in Figure 1. An excess of Xis prevents the DNA from being integrated, so it prevents lysogeny. There are two potential sets of late genes depending on whether lysis or lysogeny has been chosen. The key to this decision is the activity of the protein CII.
This protein activates the PRE promoter which gives a jump-start to the production of the protein CI. With it, positive feedback in PRM further increases CI production and locks out further Cro production driving the virus down the lysogeny pathway. The activity of CII is determined by environmental factors. Bacterial proteases enzymes that degrade proteins attack and destroy CII readily, making it very unstable.
Growth in a nutrient rich medium activates these proteases whereas starvation has the opposite effect. For this reason, well-fed cells tend towards lysis. The production of CIII is limited by the terminator switch. Therefore, higher multiplicities of infection lead to a higher probability of lysogeny. The late genes active for the lysis case are shown in Figure 1.
The protein Q that also built up during the early stage activates the PR0 promoter. As a result, genes are transcribed that code for proteins to construct the heads and tails of new viral capsids as well as those necessary to lyse the cell. The late genes for the lysogeny case are shown in Figure 1. The mRNA transcripts produced from the Pantiq promoter are complementary to the transcripts for the gene Q. Therefore, these transcripts can bind to the transcripts for the gene Q preventing them from being translated into Q proteins and helping to prevent the activation of PR0 that is needed by the lysis pathway.
The promoter PRE produces transcripts of the gene cI. The promoter PI is located in the middle of the xis gene, so the transcripts that it produces do not produce the Xis protein. Finally, this process can be reversed during induction. For Instructors Request Inspection Copy. Focusing on genetic regulatory networks, Engineering Genetic Circuits presents the modeling, analysis, and design methods for systems biology. It discusses how to examine experimental data to learn about mathematical models, develop efficient abstraction and simulation methods to analyze these models, and use analytical methods to guide the design of new circuits.
After reviewing the basic molecular biology and biochemistry principles needed to understand genetic circuits, the book describes modern experimental techniques and methods for discovering genetic circuit models from the data generated by experiments. The next four chapters present state-of-the-art methods for analyzing these genetic circuit models. The final chapter explores how researchers are beginning to use analytical methods to design synthetic genetic circuits. This text clearly shows how the success of systems biology depends on collaborations between engineers and biologists.
From biomolecular observations to mathematical models to circuit design, it provides essential information on genetic circuits and engineering techniques that can be used to study biological systems. A co-inventor on four patents and author of more than 80 technical papers and the textbook Asynchronous Circuit Design , Dr. His research interests include formal verification, asynchronous circuit design, and the analysis and design of genetic regulatory circuits. I find the many illustrations worked-out examples and ample number of figures and exercises at the end of each chapter quite useful and important.
We provide complimentary e-inspection copies of primary textbooks to instructors considering our books for course adoption. Learn More about VitalSource Bookshelf. CPD consists of any educational activity which helps to maintain and develop knowledge, problem-solving, and technical skills with the aim to provide better health care through higher standards.
It could be through conference attendance, group discussion or directed reading to name just a few examples. We provide a free online form to document your learning and a certificate for your records. Already read this title?