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?

We need more flexibility in transcriptional and translational control.

What if ...

• we had transcriptional regulators that could be used in any strain or any chassis?
• we could directly engineer repressors for target sequences, instead of having to assemble parts to place them the under control of an inducible promoter?
• we could control this repression system with a molecule of choice?
• we could increase the degrees of freedom that we as researchers have in the control of gene expression pathways?

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. 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.

The Solution

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 [1]. 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 [2]. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding [3,4]. 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 [2].

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 [5]. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding [5]. 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 [6,7]. 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 [8]. 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 the expression of any gene of interest, using wider variety of inducers than has been common to date. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop it will be possible to develop a fully orthogonal, highly versatile systems 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 applications such as developmental research. We have demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.

Project Background Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression.	Results Check out the cool results of our experiments with RiboTALs.
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.

References

[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.
[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.
[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.
[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.
[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.
[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.
[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.
[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.
[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.