Team:UC Davis/Project Overview

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In Synthetic Biology, every circuit or device contains, at its core, at least one promoter and one protein coding region.
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In Synthetic Biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar?
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<p>While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? What if we had transcriptional regulators that could be used in any strain or any chasis? What if we could directly engineer repressors for target sequences, instead of having to assemble parts to place them the under control of an inducible promoter? Furthermore, what if we could control this repression system with a molecule of choice? We would have the ability to host multiple, orthogonal systems within the same chassis. The need to 'bioprospect' metabolites would diminish. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. Finally, what if we could increase the degrees of freedom that we as researchers have in the control of gene expression pathways? If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.
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<p>What if we had transcriptional regulators that could be used in any strain or any chasis? What if we could directly engineer repressors for target sequences, instead of having to assemble parts to place them the under control of an inducible promoter? Furthermore, what if we could control this repression system with a molecule of choice? We would have the ability to host multiple, orthogonal systems within the same chassis. The need to 'bioprospect' metabolites would diminish. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. Finally, what if we could increase the degrees of freedom that we as researchers have in the control of gene expression pathways? If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.
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Revision as of 20:22, 24 September 2013

PROJECT MOTIVATION

In Synthetic Biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are countless usefully proteins we could want to create, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar?

What if we had transcriptional regulators that could be used in any strain or any chasis? What if we could directly engineer repressors for target sequences, instead of having to assemble parts to place them the under control of an inducible promoter? Furthermore, what if we could control this repression system with a molecule of choice? We would have the ability to host multiple, orthogonal systems within the same chassis. The need to 'bioprospect' metabolites would diminish. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. Finally, what if we could increase the degrees of freedom that we as researchers have in the control of gene expression pathways? If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.

PROJECT BACKGROUND

Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial pathogen Xanthomonas that contain sequence specific DNA binding domains and can act as transcriptional repressors or activators (Mahfouz et al 2012). This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, the composition of each which corresponds to a certain base preference (Meckler et al). TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding (Boch J et al 2009, Moscou et al 2009). TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices (Meckler et al 2013).


Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure (Caron et al 2012). This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding (Caron et al 2012). Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well (Topp et al 2010, Muranaka et al 2009). Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system (Dixon et al 2009). These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.


The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control, in an inducible manner, the expression of any gene of interest. Our group’s goal is to develop a demonstrate the use of TAL repressors with different theophylline riboswitches and thus develop a library of fully characterized inducible repression devices, as well as a software application capable of predicting the functionality and dynamics of a riboswitch-TALE combination. Since our control device is RNA-based, a number of transcriptional and translational steps involved in usual genetic control constructs are eliminated, reducing noise. Furthermore, as our understanding of riboswitches develops it may be possible to develop a fully orthogonal, highly versatile system for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for many applications, such as developmental research.

Project Background

Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression.

Results

Check out the results of our experiments.

Human Practices

Take a look at how we designed a new database for better raw data characterization of Biobricks.

Judging Criteria

Here's the criteria that we met for this year's team.