Team:Duke/Project/Problem

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=The ProblemDUKESUX =
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<h1> <b>Designing Synthetic Gene Networks Using Artificial <br><br>Transcription Factors in Yeast</b> </h1>
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==Genetic Toggle Switch==
 
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The Genetic Toggle Switch was among the first artificially constructed Gene Regulatory Networks. The original Genetic Toggle Switch consisted of the ptrc-2 (lacI repressible) promoter paired with the temperature sensitive lambda repressor and the PLs1con (lambda repressible) promoter paired with the lacI repressor. Subsequently, induction of the ptrc-2 promoter would repress the PLs1con promoter through expression of the lambda repressor, while induction of the PLs1con promoter would repress the ptrc-2 promoter through expression of the lacI repressor. Switching of the toggle switch from one promoter to another was accomplished by addition of either Isopropyl β-D-1-thiogalactopyranoside (IPTG), which causes the lacI to unbind allowing the ptrc-2 to switch on, or a thermal pulse, which causes the lambda repressor to unbind and allow the PLs1con promoter to switch on. In order to characterize the toggle switch, a GFPmut3 structural gene was placed downstream of the Ptrc-2 promoter so that induction of the Ptrc-2 promoter would result in high expression of GFPmut3 while induction of PLs1con would result in low expression of GFPmut3.
 
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[[File:DukeToggleSwitch.png|center|frame|Figure 1. Genetic Toggle Switch]]
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=Project Description=
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Following an initial period of growth after the publication of the first synthetic gene circuits, development in the field has stalled. This is due in part to the limited number of well-characterized parts with desired features. For instance, the function of the repressilator and genetic toggle switch both rely on repressible promoters with high cooperativity – provided in these cases by multimerization of the repressor proteins. The TALE family of transcription factors (TFs) and the CRISPR/Cas9 system show promise in expanding the parts list to bind to near-arbitrary target sequences, but because they bind to DNA as monomers, promoters under their control cannot show cooperativity in their response. 
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==Characteristics of Genetic Toggle Switches==
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In general, Genetic Toggle Switches have several characteristics which make them unique from other Gene Regulatory networks. For example, Genetic Toggle Switches must be capable of bi-stable behavior, meaning that they should only exist in one of two stable states instead of in a variety of intermediate states. Bi-stability also suggests that a certain threshold must be passed before the toggle switch can switch from one state to another. In addition, toggle switches should have low basal transcriptional noise to prevent random switching without outside induction. Finally, a reporter or marker structural gene, such as a fluorescent protein, should be present in order to characterize the effectiveness of the toggle switch.
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It has been shown theoretically and in vivo that repressors binding as monomers to multiple binding sites can introduce cooperativity in to a system. With this in mind, we are developing an organism-independent approach that leverages programmable TFs to create library of independent and orthogonal repressor-promoter pairs with a range of expression parameters (viz. cooperativity, basal and maximal expression rate, response time) of potentially unlimited size. It is our aim that this approach will enable the field to move toward exploring higher-order dynamics.
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**This is from 2011 Team Duke**
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=Abstract=
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Synthetic gene circuits have the potential to revolutionize gene therapies and bio-industrial methods by allowing predictable, customized control of gene expression. Bistable switches and oscillators, key building blocks of more complex gene networks, have been constructed using naturally occurring and well-characterized regulatory elements. In order to expand the versatility and variety of these circuits, we designed and constructed gene networks using artificial transcription factors (ATFs). The ATFs are of two classes: inhibitory TAL proteins and a catalytically inactive dCas9 protein with small guide RNA elements, each orthogonal to the yeast genome. Using mathematical modeling, we determined the parameters expected to create bistability and oscillation, using tandem binding site kinetics to achieve cooperativity. Based on these results, we assembled a library of plasmids containing ATFs, binding sites, regulatory elements, and fluorescent reporters. We then integrated these genes into the genome of <i>Saccharomyces cerevisiae</i> and are currently characterizing them using flow cytometry.
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== Building a Toggle Switch in Fission Yeast ==
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=References=
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#Gardener,T. et al. Construction of a genetic toggle switch in Escherichia coli. Nature. 403, 339-342 (2000).
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Latest revision as of 06:43, 27 September 2013

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Designing Synthetic Gene Networks Using Artificial

Transcription Factors in Yeast


Project Description

Following an initial period of growth after the publication of the first synthetic gene circuits, development in the field has stalled. This is due in part to the limited number of well-characterized parts with desired features. For instance, the function of the repressilator and genetic toggle switch both rely on repressible promoters with high cooperativity – provided in these cases by multimerization of the repressor proteins. The TALE family of transcription factors (TFs) and the CRISPR/Cas9 system show promise in expanding the parts list to bind to near-arbitrary target sequences, but because they bind to DNA as monomers, promoters under their control cannot show cooperativity in their response.

It has been shown theoretically and in vivo that repressors binding as monomers to multiple binding sites can introduce cooperativity in to a system. With this in mind, we are developing an organism-independent approach that leverages programmable TFs to create library of independent and orthogonal repressor-promoter pairs with a range of expression parameters (viz. cooperativity, basal and maximal expression rate, response time) of potentially unlimited size. It is our aim that this approach will enable the field to move toward exploring higher-order dynamics.


Abstract

Synthetic gene circuits have the potential to revolutionize gene therapies and bio-industrial methods by allowing predictable, customized control of gene expression. Bistable switches and oscillators, key building blocks of more complex gene networks, have been constructed using naturally occurring and well-characterized regulatory elements. In order to expand the versatility and variety of these circuits, we designed and constructed gene networks using artificial transcription factors (ATFs). The ATFs are of two classes: inhibitory TAL proteins and a catalytically inactive dCas9 protein with small guide RNA elements, each orthogonal to the yeast genome. Using mathematical modeling, we determined the parameters expected to create bistability and oscillation, using tandem binding site kinetics to achieve cooperativity. Based on these results, we assembled a library of plasmids containing ATFs, binding sites, regulatory elements, and fluorescent reporters. We then integrated these genes into the genome of Saccharomyces cerevisiae and are currently characterizing them using flow cytometry.