Team:Debrecen-Hungary/project

From 2010.igem.org

(Difference between revisions)
(Introduction)
(Introduction)
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The international genetically engineered machines and associated parts registry are, to date, one of the largest registries for standard parts in use for synthetic biology. Free information can be found in the registry regarding parts, devices and modules all inputted by various teams worldwide.
The international genetically engineered machines and associated parts registry are, to date, one of the largest registries for standard parts in use for synthetic biology. Free information can be found in the registry regarding parts, devices and modules all inputted by various teams worldwide.
-
Animal synthetic biology is still in its infancy. This large kingdom includes all multicellular eukaryotes such as mammalians, arthropods and nematodes. No standard chassis (framework) exists for the animal kingdom which makes them far less popular then the famous E.coli. Very few iGEM teams (or even labs outside iGEM) have chosen to toggle the animal chassis (two notable examples are team Heidelberg 2009 and team Slovenia 2006).    The amount of available compatible parts is limited, which severely restricts the options of creating complex biological devices. Nearly no imagination is required for designing tools, since their analogues already exist in the bacterial chassis. The possible use of such systems is unlimited. Field’s such as of environment, medicine, energy and research all gain to profit from the development of animal synthetic biology.
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[http://2010.igem.org/Team:Debrecen-Hungary/minimals#Drosophila_Melanogaster Animal synthetic biology] is still in its infancy. This large kingdom includes all multicellular eukaryotes such as mammalians, arthropods and nematodes. No standard chassis (framework) exists for the animal kingdom which makes them far less popular then the famous E.coli. Very few iGEM teams (or even labs outside iGEM) have chosen to toggle the animal chassis (two notable examples are team Heidelberg 2009 and team Slovenia 2006).    The amount of available compatible parts is limited, which severely restricts the options of creating complex biological devices. Nearly no imagination is required for designing tools, since their analogues already exist in the bacterial chassis. The possible use of such systems is unlimited. Field’s such as of environment, medicine, energy and research all gain to profit from the development of animal synthetic biology.
Systems requiring gene expression input in animal synthetic biology systems require a way to standardize gene expression, a complicated task. The way from gene to protein contains many steps of possible error: transcription factor binding, promoter strength, recruitment of auxiliary proteins, nuclear RNA synthesis and many more steps finally leading to translation, folding, cleaving and delivery (but hey, you have to start somewhere).  
Systems requiring gene expression input in animal synthetic biology systems require a way to standardize gene expression, a complicated task. The way from gene to protein contains many steps of possible error: transcription factor binding, promoter strength, recruitment of auxiliary proteins, nuclear RNA synthesis and many more steps finally leading to translation, folding, cleaving and delivery (but hey, you have to start somewhere).  
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Our team was interested at designing animal synthetic biology tools related to PoPs. PoPs (polymerase per second), the flying Dutchman of synthetic biology, is a number which represents the rate (base pair per second) at which RNA polymerase crosses past a given DNA position. Currently, no in vivo technique for measuring PoPS directly exists; it can be estimated indirectly by measuring other parameters (eg protein expression or enzyme activity). Nevertheless it is still a useful abstraction for thinking about transcription-based logic devices and it allows the engineer to define devices. Our aim, was not only to infer PoPs but to devise a way to titrate it remotely.
Our team was interested at designing animal synthetic biology tools related to PoPs. PoPs (polymerase per second), the flying Dutchman of synthetic biology, is a number which represents the rate (base pair per second) at which RNA polymerase crosses past a given DNA position. Currently, no in vivo technique for measuring PoPS directly exists; it can be estimated indirectly by measuring other parameters (eg protein expression or enzyme activity). Nevertheless it is still a useful abstraction for thinking about transcription-based logic devices and it allows the engineer to define devices. Our aim, was not only to infer PoPs but to devise a way to titrate it remotely.
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Nuclear receptors, best viewed as transcription factors which can be activated by extracellular cues, are unique in their ability to allow direct remote PoPs titration through extracellular cues (both activation and repression). These receptor classes bears high homology to each other throughout the animal kingdom and are modular into distinct domains. All of these features attracted our attention to find a way to incorporate these tools in the parts registry, and characterize them in standardized methods.
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[http://2010.igem.org/Team:Debrecen-Hungary/minimals#Nuclear_Receptos Nuclear receptors], best viewed as transcription factors which can be activated by extracellular cues, are unique in their ability to allow direct remote PoPs titration (both activation and repression). These receptor classes bears high homology to each other throughout the animal kingdom and are modular into distinct domains. All of these features attracted our attention to find a way to incorporate these tools in the parts registry, and characterize them in standardized methods.
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The ligand binding domains (LBD) are the segment of the receptor which changes its conformation upon lipophilic ligand binding; this also causes the exposure of a powerful transcriptional activation domain (which attracts the transcriptional machinery).  This was our segment of interest since it links ligand binding with transcriptional activity.  
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[http://2010.igem.org/Team:Debrecen-Hungary/minimals#Ligand_binding_domains The ligand binding domains (LBD)] are the segment of the receptor which changes its conformation upon lipophilic ligand binding; this also causes the exposure of a powerful transcriptional activation domain (which attracts the transcriptional machinery).  This was our segment of interest since it links ligand binding with transcriptional activity.  
Our team has generated a library of ligand binding domains for the rational construction of synthetic titrateable transcription factors.  In our model hybrid receptor, this LBD was fused to Gal4 a DNA binding domain.  The sequance to which Gal4 binds is CGG-N11-CCG, where N can be any base[3]. Although Gal4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription factor in a variety of organisms such as Drosophila[4], and human cells[5], highlighting that the same mechanisms for gene expression have been conserved over the course of evolution.  
Our team has generated a library of ligand binding domains for the rational construction of synthetic titrateable transcription factors.  In our model hybrid receptor, this LBD was fused to Gal4 a DNA binding domain.  The sequance to which Gal4 binds is CGG-N11-CCG, where N can be any base[3]. Although Gal4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription factor in a variety of organisms such as Drosophila[4], and human cells[5], highlighting that the same mechanisms for gene expression have been conserved over the course of evolution.  
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The examination of these chimeric receptors was through a version of the two hybrid hypothesis. We moved these composite parts into an expression vector and transfected them into COS- 1 cells.  The new “hybrid” receptors were designed to enhance the gene expression of a luciferase enzyme through a GAL4 sensitive promoter (which was also transfected).  The luciferase activity was assessed photometrically for several ligand concentrations and normalized to a constitutively expressed beta-gal activity. The final result was plotted on a dose response curve. The EC50 was then extracted from the curve.
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The examination of these chimeric receptors was through a version of the [http://2010.igem.org/Team:Debrecen-Hungary/minimals#Two_Hybrid_Screening two hybrid screeining]. We moved these composite parts into an expression vector and transfected them into [http://2010.igem.org/Team:Debrecen-Hungary/minimals#Cos-1_cells  COS- 1 cells].  The new “hybrid” receptors were designed to enhance the gene expression of a luciferase enzyme through a GAL4 sensitive promoter (which was also transfected).  The [http://2010.igem.org/Team:Debrecen-Hungary/minimals#Luciferase luciferase activity] was assessed photometrically for several ligand concentrations and normalized to a constitutively expressed beta-gal activity. The final result was plotted on a [http://2010.igem.org/Team:Debrecen-Hungary/minimals#Dose_response_curve dose response curve]. The EC50 was then extracted from the curve.
The remotely activated transcription factor concept can be used to construct highly complex synthetic biological systems, for us it was a very appealing concept. Physicians may use fruit fly hormones in humans to titrate specific genes in gene therapy patient in selected tissue, such as dopamine receptor genes in schizophrenia. Worm species that can synthesize a different color reaction based on the amount of environmental pollutants in the soil they live. Scientists may induce stem cell pluripotency by titrating the exact amount of oncogenes needed for a fibroblast to turn into an embryo.
The remotely activated transcription factor concept can be used to construct highly complex synthetic biological systems, for us it was a very appealing concept. Physicians may use fruit fly hormones in humans to titrate specific genes in gene therapy patient in selected tissue, such as dopamine receptor genes in schizophrenia. Worm species that can synthesize a different color reaction based on the amount of environmental pollutants in the soil they live. Scientists may induce stem cell pluripotency by titrating the exact amount of oncogenes needed for a fibroblast to turn into an embryo.

Revision as of 10:50, 19 October 2010



Abstract

Animal synthetic biology has huge potential, yet it is still in need of more diverse molecular tools for defined gene regulation. Nuclear receptors are a conserved family of proteins responsible for sensing lipids; they may be viewed as lipid activated transcription factors. We have successfully developed a kit with a variety of lipid responsive domains (from H.sapines, D.melanogaster and C.elegans) for the rational construction of synthetic transcription factors. The domains respond only to predefined lipids and selectively activate predetermined gene expression. To characterize theses domains, we used standardized protocols for comparable measurements. In vivo gene expression was measured as a function of ligand concentration using luciferase activity. The potential for these tools is immense; e.g. from the ultra sensitive detection of lipid contaminants in the environment to the opportunity of titration specific gene expression canges in patients undergoing gene therapy.


Introduction

Synthetic biology is a relatively new area of biological research; similar to many other new scientific fields it has many definitions. The best way to express the meaning of synthetic biology is to understand the desired end result: engineering of complex biological systems. These systems are best thought of as analogues of everyday machinery: cogwheels, levers, timers, button and buzzers (in this case a clock), only in the case of biological systems (molecular ones) cells, DNA, proteins, lipids, sugars and RNA are the “parts” of the system.

Similar to mechanical engineering (or every other engineering branch) there is a need for standards (consensus way of doing things), abstraction (simple and unified way of thinking about the parts of a system), and modularity (how these parts interact to become the device, or several devices into a system). Thus a good definition for synthetic biology could be engineering of molecular (for the time being) biological systems according to preset standard parts. The international genetically engineered machines and associated parts registry are, to date, one of the largest registries for standard parts in use for synthetic biology. Free information can be found in the registry regarding parts, devices and modules all inputted by various teams worldwide.

Animal synthetic biology is still in its infancy. This large kingdom includes all multicellular eukaryotes such as mammalians, arthropods and nematodes. No standard chassis (framework) exists for the animal kingdom which makes them far less popular then the famous E.coli. Very few iGEM teams (or even labs outside iGEM) have chosen to toggle the animal chassis (two notable examples are team Heidelberg 2009 and team Slovenia 2006). The amount of available compatible parts is limited, which severely restricts the options of creating complex biological devices. Nearly no imagination is required for designing tools, since their analogues already exist in the bacterial chassis. The possible use of such systems is unlimited. Field’s such as of environment, medicine, energy and research all gain to profit from the development of animal synthetic biology.

Systems requiring gene expression input in animal synthetic biology systems require a way to standardize gene expression, a complicated task. The way from gene to protein contains many steps of possible error: transcription factor binding, promoter strength, recruitment of auxiliary proteins, nuclear RNA synthesis and many more steps finally leading to translation, folding, cleaving and delivery (but hey, you have to start somewhere).

Our team was interested at designing animal synthetic biology tools related to PoPs. PoPs (polymerase per second), the flying Dutchman of synthetic biology, is a number which represents the rate (base pair per second) at which RNA polymerase crosses past a given DNA position. Currently, no in vivo technique for measuring PoPS directly exists; it can be estimated indirectly by measuring other parameters (eg protein expression or enzyme activity). Nevertheless it is still a useful abstraction for thinking about transcription-based logic devices and it allows the engineer to define devices. Our aim, was not only to infer PoPs but to devise a way to titrate it remotely.

Nuclear receptors, best viewed as transcription factors which can be activated by extracellular cues, are unique in their ability to allow direct remote PoPs titration (both activation and repression). These receptor classes bears high homology to each other throughout the animal kingdom and are modular into distinct domains. All of these features attracted our attention to find a way to incorporate these tools in the parts registry, and characterize them in standardized methods.

The ligand binding domains (LBD) are the segment of the receptor which changes its conformation upon lipophilic ligand binding; this also causes the exposure of a powerful transcriptional activation domain (which attracts the transcriptional machinery). This was our segment of interest since it links ligand binding with transcriptional activity.

Our team has generated a library of ligand binding domains for the rational construction of synthetic titrateable transcription factors. In our model hybrid receptor, this LBD was fused to Gal4 a DNA binding domain. The sequance to which Gal4 binds is CGG-N11-CCG, where N can be any base[3]. Although Gal4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription factor in a variety of organisms such as Drosophila[4], and human cells[5], highlighting that the same mechanisms for gene expression have been conserved over the course of evolution.

The examination of these chimeric receptors was through a version of the two hybrid screeining. We moved these composite parts into an expression vector and transfected them into COS- 1 cells. The new “hybrid” receptors were designed to enhance the gene expression of a luciferase enzyme through a GAL4 sensitive promoter (which was also transfected). The luciferase activity was assessed photometrically for several ligand concentrations and normalized to a constitutively expressed beta-gal activity. The final result was plotted on a dose response curve. The EC50 was then extracted from the curve.

The remotely activated transcription factor concept can be used to construct highly complex synthetic biological systems, for us it was a very appealing concept. Physicians may use fruit fly hormones in humans to titrate specific genes in gene therapy patient in selected tissue, such as dopamine receptor genes in schizophrenia. Worm species that can synthesize a different color reaction based on the amount of environmental pollutants in the soil they live. Scientists may induce stem cell pluripotency by titrating the exact amount of oncogenes needed for a fibroblast to turn into an embryo.










Our team has decided to focus efforts at making a nuclear hormone receptor kit, for use in eukaryotic cells. Our attention was drawn to this class of receptors due to their key physiological contribution in the endocrine system, cellular differentiation pathways, paracrine signaling and more.

 Upon ligand binding, nuclear hormone receptors undergo a change in conformation. They then translocate to the nucleus where they bind to specific DNA elements. The binding ultimately leads to defined changes in gene expression (both activation and repression). They may be viewed as special transcription factors which can be activated by extracellular cues.

The kit will include various basic parts of which a user might be able to create his own ligand activated transcription factor (a composite part). Our primary goal is thorough characterization of each basic part. Our methods and results as well as secondary goals will be addressed at a later time.

We hope that our biobrick part contribution may be used in a variety of medicinal applications as well as environmental sustainability projects or even other fields.