Synthetic biological circuit

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The lac operon is a natural biological circuit on which many synthetic circuits are based. Top: Repressed, Bottom: Active. 1: RNA polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA. Lac Operon.svg
The lac operon is a natural biological circuit on which many synthetic circuits are based. Top: Repressed, Bottom: Active.
1: RNA polymerase, 2: Repressor, 3: Promoter, 4: Operator, 5: Lactose, 6: lacZ, 7: lacY, 8: lacA.

Synthetic biological circuits are an application of synthetic biology where biological parts inside a cell are designed to perform logical functions mimicking those observed in electronic circuits. Typically, these circuits are categorized as either genetic circuits, RNA circuits, or protein circuits, depending on the types of biomolecule that interact to create the circuit's behavior. The applications of all three types of circuit range from simply inducing production to adding a measurable element, like green fluorescent protein, to an existing natural biological circuit, to implementing completely new systems of many parts. [1]

Contents

A ribosome is a biological machine. Protein translation.gif
A ribosome is a biological machine.

The goal of synthetic biology is to generate an array of tunable and characterized parts, or modules, with which any desirable synthetic biological circuit can be easily designed and implemented. [2] These circuits can serve as a method to modify cellular functions, create cellular responses to environmental conditions, or influence cellular development. By implementing rational, controllable logic elements in cellular systems, researchers can use living systems as engineered "biological machines" to perform a vast range of useful functions. [1]

History

The first natural gene circuit studied in detail was the lac operon. In studies of diauxic growth of E. coli on two-sugar media, Jacques Monod and Francois Jacob discovered that E.coli preferentially consumes the more easily processed glucose before switching to lactose metabolism. They discovered that the mechanism that controlled the metabolic "switching" function was a two-part control mechanism on the lac operon. When lactose is present in the cell the enzyme β-galactosidase is produced to convert lactose into glucose or galactose. When lactose is absent in the cell the lac repressor inhibits the production of the enzyme β-galactosidase to prevent any inefficient processes within the cell.

The lac operon is used in the biotechnology industry for production of recombinant proteins for therapeutic use. The gene or genes for producing an exogenous protein are placed on a plasmid under the control of the lac promoter. Initially the cells are grown in a medium that does not contain lactose or other sugars, so the new genes are not expressed. Once the cells reach a certain point in their growth, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added. IPTG, a molecule similar to lactose, but with a sulfur bond that is not hydrolyzable so that E. coli does not digest it, is used to activate or "induce" the production of the new protein. Once the cells are induced, it is difficult to remove IPTG from the cells and therefore it is difficult to stop expression.

Two early examples of synthetic biological circuits were published in Nature in 2000. One, by Tim Gardner, Charles Cantor, and Jim Collins working at Boston University, demonstrated a "bistable" switch in E. coli. The switch is turned on by heating the culture of bacteria and turned off by addition of IPTG. They used green fluorescent protein as a reporter for their system. [3] The second, by Michael Elowitz and Stanislas Leibler, showed that three repressor genes could be connected to form a negative feedback loop termed the Repressilator that produces self-sustaining oscillations of protein levels in E. coli. [4]

Currently, synthetic circuits are a burgeoning area of research in systems biology with more publications detailing synthetic biological circuits published every year. [5] There has been significant interest in encouraging education and outreach as well: the International Genetically Engineered Machines Competition [6] manages the creation and standardization of BioBrick parts as a means to allow undergraduate and high school students to design their own synthetic biological circuits.

Interest and goals

Both immediate and long term applications exist for the use of synthetic biological circuits, including different applications for metabolic engineering, and synthetic biology. Those demonstrated successfully include pharmaceutical production, [7] and fuel production. [8] However, methods involving direct genetic introduction are not inherently effective without invoking the basic principles of synthetic cellular circuits. For example, each of these successful systems employs a method to introduce all-or-none induction or expression. This is a biological circuit where a simple repressor or promoter is introduced to facilitate creation of the product, or inhibition of a competing pathway. However, with the limited understanding of cellular networks and natural circuitry, implementation of more robust schemes with more precise control and feedback is hindered. Therein lies the immediate interest in synthetic cellular circuits.

Development in understanding cellular circuitry can lead to exciting new modifications, such as cells which can respond to environmental stimuli. For example, cells could be developed that signal toxic surroundings and react by activating pathways used to degrade the perceived toxin. [9] To develop such a cell, it is necessary to create a complex synthetic cellular circuit which can respond appropriately to a given stimulus.

Given synthetic cellular circuits represent a form of control for cellular activities, it can be reasoned that with complete understanding of cellular pathways, "plug and play" [1] cells with well defined genetic circuitry can be engineered. It is widely believed that if a proper toolbox of parts is generated, [10] synthetic cells can be developed implementing only the pathways necessary for cell survival and reproduction. From this cell, to be thought of as a minimal genome cell, one can add pieces from the toolbox to create a well defined pathway with appropriate synthetic circuitry for an effective feedback system. Because of the basic ground up construction method, and the proposed database of mapped circuitry pieces, techniques mirroring those used to model computer or electronic circuits can be used to redesign cells and model cells for easy troubleshooting and predictive behavior and yields.

Example circuits

Oscillators

  1. Repressilator
  2. Mammalian tunable synthetic oscillator
  3. Bacterial tunable synthetic oscillator
  4. Coupled bacterial oscillator
  5. Globally coupled bacterial oscillator

Elowitz et al. and Fung et al. created oscillatory circuits that use multiple self-regulating mechanisms to create a time-dependent oscillation of gene product expression. [11] [12]

Bistable switches

  1. Toggle-switch

Gardner et al. used mutual repression between two control units to create an implementation of a toggle switch capable of controlling cells in a bistable manner: transient stimuli resulting in persistent responses. [3]

Gene regulation is an essential part of developmental processes. During development, genes are turned on and off in different tissues, changes in regulatory mechanisms may result in genetic switching in a bistable system, the gene switches serve as regulatory molecule binding sites. These are proteins that activate transcription when they land on a gene switch and thereby express the gene that was expected to operate as a memory device, allowing cell fate decisions to be chosen and maintained. [13]

Toggle switch which operates using two mutually inhibitory genes, each promoter is inhibited by the repressor that is transcribed by the opposing promoter. Toggle switch design: Inducer 1 inactivates repressor 1, which means repressor 2 is produced. Repressor 2, in turn, stops transcription of the repressor 1 gene and the reporter gene. [14]

Logical operators

The logical AND gate. If Signal A AND Signal B are present, then the desired gene product will result. All promoters shown are inducible, activated by the displayed gene product. Each signal activates expression of a separate gene (shown in light blue). The expressed proteins then can either form a complete complex in cytosol, that is capable of activating expression of the output (shown), or can act separately to induce expression, such as separately removing an inhibiting protein and inducing activation of the uninhibited promoter. SynBioCirc-AndLogicGate.jpg
The logical AND gate. If Signal A AND Signal B are present, then the desired gene product will result. All promoters shown are inducible, activated by the displayed gene product. Each signal activates expression of a separate gene (shown in light blue). The expressed proteins then can either form a complete complex in cytosol, that is capable of activating expression of the output (shown), or can act separately to induce expression, such as separately removing an inhibiting protein and inducing activation of the uninhibited promoter.
The logical OR gate. If Signal A OR Signal B are present, then the desired gene product will result. All promoters shown are inducible. Either signal is capable of activating the expression of the output gene product, and only the action of a single promoter is required for gene expression. Post-transcriptional regulation mechanisms can prevent the presence of both inputs producing a compounded high output, such as implementing a low binding affinity ribosome binding site. SynBioCirc-OrLogicGate.jpg
The logical OR gate. If Signal A OR Signal B are present, then the desired gene product will result. All promoters shown are inducible. Either signal is capable of activating the expression of the output gene product, and only the action of a single promoter is required for gene expression. Post-transcriptional regulation mechanisms can prevent the presence of both inputs producing a compounded high output, such as implementing a low binding affinity ribosome binding site.
The logical Negated AND gate. If Signal A AND Signal B are present, then the desired gene product will NOT result. All promoters shown are inducible. The activating promoter for the output gene is constitutive, and thus not shown. The constitutive promoter for the output gene keeps it "on" and is only deactivated when (similar to the AND gate) a complex as a result of two input signal gene products blocks the expression of the output gene. SynBioCirc-NandLogicGate.jpg
The logical Negated AND gate. If Signal A AND Signal B are present, then the desired gene product will NOT result. All promoters shown are inducible. The activating promoter for the output gene is constitutive, and thus not shown. The constitutive promoter for the output gene keeps it "on" and is only deactivated when (similar to the AND gate) a complex as a result of two input signal gene products blocks the expression of the output gene.

Analog tuners

Using negative feedback and identical promoters, linearizer gene circuits can impose uniform gene expression that depends linearly on extracellular chemical inducer concentration. [17]

Controllers of gene expression heterogeneity

Synthetic gene circuits can control gene expression heterogeneity can be controlled independently of the gene expression mean. [18]

Other engineered systems

Engineered systems are the result of implementation of combinations of different control mechanisms. A limited counting mechanism was implemented by a pulse-controlled gene cascade [19] and application of logic elements enables genetic "programming" of cells as in the research of Tabor et al., which synthesized a photosensitive bacterial edge detection program. [20]

Circuit design

Computational design and evaluation of DNA circuits to achieve optimal performance Computational design and evaluation of DNA circuits to achieve optimal performance.svg
Computational design and evaluation of DNA circuits to achieve optimal performance

Recent developments in artificial gene synthesis and the corresponding increase in competition within the industry have led to a significant drop in price and wait time of gene synthesis and helped improve methods used in circuit design. [21] At the moment, circuit design is improving at a slow pace because of insufficient organization of known multiple gene interactions and mathematical models. This issue is being addressed by applying computer-aided design (CAD) software to provide multimedia representations of circuits through images, text and programming language applied to biological circuits. [22] Some of the more well known CAD programs include GenoCAD, Clotho framework and j5. [23] [24] [25] GenoCAD uses grammars, which are either opensource or user generated "rules" which include the available genes and known gene interactions for cloning organisms. Clotho framework uses the Biobrick standard rules. [22]

Related Research Articles

<span class="mw-page-title-main">Bistability</span> Quality of a system having two stable equilibrium states

In a dynamical system, bistability means the system has two stable equilibrium states. A bistable structure can be resting in either of two states. An example of a mechanical device which is bistable is a light switch. The switch lever is designed to rest in the "on" or "off" position, but not between the two. Bistable behavior can occur in mechanical linkages, electronic circuits, nonlinear optical systems, chemical reactions, and physiological and biological systems.

In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splicing to create monocistronic mRNAs that are translated separately, i.e. several strands of mRNA that each encode a single gene product. The result of this is that the genes contained in the operon are either expressed together or not at all. Several genes must be co-transcribed to define an operon.

<span class="mw-page-title-main">François Jacob</span> French biologist

François Jacob was a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through regulation of transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.

<span class="mw-page-title-main">Gene regulatory network</span> Collection of molecular regulators

A generegulatory network (GRN) is a collection of molecular regulators that interact with each other and with other substances in the cell to govern the gene expression levels of mRNA and proteins which, in turn, determine the function of the cell. GRN also play a central role in morphogenesis, the creation of body structures, which in turn is central to evolutionary developmental biology (evo-devo).

<span class="mw-page-title-main">Lac repressor</span> DNA-binding protein

The lac repressor (LacI) is a DNA-binding protein that inhibits the expression of genes coding for proteins involved in the metabolism of lactose in bacteria. These genes are repressed when lactose is not available to the cell, ensuring that the bacterium only invests energy in the production of machinery necessary for uptake and utilization of lactose when lactose is present. When lactose becomes available, it is firstly converted into allolactose by β-Galactosidase (lacZ) in bacteria. The DNA binding ability of lac repressor bound with allolactose is inhibited due to allosteric regulation, thereby genes coding for proteins involved in lactose uptake and utilization can be expressed.

<i>lac</i> operon Set genes encoding proteins and enzymes for lactose metabolism

The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.

<span class="mw-page-title-main">Synthetic biology</span> Interdisciplinary branch of biology and engineering

Synthetic biology (SynBio) is a multidisciplinary field of science that focuses on living systems and organisms, and it applies engineering principles to develop new biological parts, devices, and systems or to redesign existing systems found in nature.

A circadian clock, or circadian oscillator, also known as one’s internal alarm clock is a biochemical oscillator that cycles with a stable phase and is synchronized with solar time.

<span class="mw-page-title-main">Silencer (genetics)</span> Type of DNA sequence

In genetics, a silencer is a DNA sequence capable of binding transcription regulation factors, called repressors. DNA contains genes and provides the template to produce messenger RNA (mRNA). That mRNA is then translated into proteins. When a repressor protein binds to the silencer region of DNA, RNA polymerase is prevented from transcribing the DNA sequence into RNA. With transcription blocked, the translation of RNA into proteins is impossible. Thus, silencers prevent genes from being expressed as proteins.

<span class="mw-page-title-main">René Thomas (biologist)</span>

René Thomas (14 May 1928 - 9 January 2017 was a Belgian scientist. His research included DNA biochemistry and biophysics, genetics, mathematical biology, and finally dynamical systems. He devoted his life to the deciphering of key logical principles at the basis of the behaviour of biological systems, and more generally to the generation of complex dynamical behaviour. He was professor and laboratory head at the Université Libre de Bruxelles, and taught and inspired several generations of researchers.

The repressilator is a genetic regulatory network consisting of at least one feedback loop with at least three genes, each expressing a protein that represses the next gene in the loop. In biological research, repressilators have been used to build cellular models and understand cell function. There are both artificial and naturally-occurring repressilators. Recently, the naturally-occurring repressilator clock gene circuit in Arabidopsis thaliana and mammalian systems have been studied.

<span class="mw-page-title-main">James J. Collins</span> American bioengineer

James Joseph Collins is an American biomedical engineer and bioengineer who serves as the Termeer Professor of Medical Engineering & Science at the Massachusetts Institute of Technology (MIT), where he is also a director at the MIT Abdul Latif Jameel Clinic for Machine Learning in Health.

<span class="mw-page-title-main">Christopher Voigt</span> American bioengineer

Christopher Voigt is an American synthetic biologist, molecular biophysicist, and engineer.

cAMP responsive element modulator Protein-coding gene in the species Homo sapiens

cAMP responsive element modulator is a protein that in humans is encoded by the CREM gene, and it belongs to the cAMP-responsive element binding protein family. It has multiple isoforms, which act either as repressors or activators. CREB family is important for in regulating transcription in response to various stresses, metabolic and developmental signals. CREM transcription factors also play an important role in many physiological systems, such as cardiac function, circadian rhythms, locomotion and spermatogenesis.

Michael B. Elowitz is a biologist and professor of Biology, Bioengineering, and Applied Physics at the California Institute of Technology, and investigator at the Howard Hughes Medical Institute. In 2007 he was the recipient of the Genius grant, better known as the MacArthur Fellows Program for the design of a synthetic gene regulatory network, the Repressilator, which helped initiate the field of synthetic biology. He was the first to show how inherently random effects, or 'noise', in gene expression could be detected and quantified in living cells, leading to a growing recognition of the many roles that noise plays in living cells. His work in Synthetic Biology and Noise represent two foundations of the field of Systems Biology. Since then, his laboratory has contributed to the development of synthetic biological circuits that perform a range of functions inside cells, and revealed biological circuit design principles underlying epigenetic memory, cell fate control, cell-cell communication, and multicellular behaviors.

Johan Paulsson is a Swedish mathematician and systems biologist at Harvard Medical School. He is a leading researcher in systems biology and stochastic processes, specializing in stochasticity in gene networks and plasmid reproduction.

Wendell Lim is an American biochemist who is the Byer's Distinguished Professor of Cellular and Molecular Pharmacology at the University of California, San Francisco. He is the director of the UCSF Cell Design Institute. He earned his A.B. in chemistry from Harvard University working with Jeremy Knowles on enzyme evolutionary optimization. He obtained his Ph.D in biochemistry and biophysics from Massachusetts Institute of Technology under the guidance of Bob Sauer using genetic and biophysical approaches to understand the role of hydrophobic core interactions in protein folding. He then did his postdoctoral work with Frederic Richards at Yale University on the structure of protein interaction domains. Lim's work has focused on cell signaling, synthetic biology, and cell engineering, particularly in immune cells.

Stanislas Leibler is a French-American theoretical and experimental biologist and physicist. He is Systems Biology Professor at the Institute for Advanced Study in Princeton and the Gladys T. Perkin Professor and Head of the Laboratory of Living Matter at the Rockefeller University.

<span class="mw-page-title-main">Gene regulatory circuit</span>

Genetic regulatory circuits is a concept that evolved from the Operon Model discovered by François Jacob and Jacques Monod. They are functional clusters of genes that impact each other's expression through inducible transcription factors and cis-regulatory elements.

The T7 expression system is used in the field of microbiology to clone recombinant DNA using strains of E. coli. It is the most popular system for expressing recombinant proteins in E. coli.

References

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  2. ↑ "Synthetic Biology: FAQ". SyntheticBiology.org. Archived from the original on 12 December 2002. Retrieved 21 December 2011.
  3. 1 2 Gardner TS, Cantor CR, Collins JJ (January 2000). "Construction of a genetic toggle switch in Escherichia coli". Nature. 403 (6767): 339–42. Bibcode:2000Natur.403..339G. doi:10.1038/35002131. PMID   10659857. S2CID   345059.
  4. ↑ Elowitz MB, Leibler S (January 2000). "A synthetic oscillatory network of transcriptional regulators". Nature. 403 (6767): 335–338. Bibcode:2000Natur.403..335E. doi:10.1038/35002125. PMID   10659856. S2CID   41632754.
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  6. ↑ International Genetically Engineered Machines (iGem) http://igem.org/Main_Page
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  23. ↑ "GenoCAD: CAD Software for Synthetic Biology". www.genocad.com. Archived from the original on 5 February 2016. Retrieved 2015-10-21.
  24. ↑ "Clotho". www.clothocad.org. Archived from the original on 26 December 2014. Retrieved 2015-10-21.
  25. ↑ "j5 automated DNA assembly". j5.jbei.org. Retrieved 2015-10-21.
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