Improving a Synthetic Riboswitch that Responds to an Herbicide Open Access

Longo, Julie Michelle (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/9z903066s?locale=en
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Abstract

Abstract
Improving a Synthetic Riboswitch that Responds to an Herbicide
By Julie Longo
A riboswitch is a part of an mRNA molecule that controls gene expression through binding to a small molecule ligand. Riboswitches are found in the 5' untranslated region of mRNA and are made up of two components, the aptamer, which binds to a small molecule ligand, and the expression platform, which changes conformation in response to the ligand. Because riboswitches influence gene expression, they can be used to reprogram bacteria to perform novel functions. The Gallivan lab recently identified a riboswitch that recognizes atrazine, a harmful herbicide. However, this switch shows residual gene expression in the absence of the ligand and only a five fold activation ratio in the presence of the ligand. This project seeks to improve this riboswitch to give it a greater dynamic range, the difference in gene expression in the presence and absence of the ligand. Methods used to alter the switching ability of the original atrazine riboswitch include randomization of a section of the expression platform and deletion of fifteen nucleotides downstream of the aptamer. By designing a better switch, it will be possible to create bacteria that will be better able to metabolize residual atrazine in the environment.

Table of Contents

Table of Contents
Introduction...1
Previous Work...5
Improving the Atrazine Riboswitch...10
Experimental...14

General Considerations...14
Library Construction...14
Screening via bacterial migration experiments...17
Screening via β-galactosidase assays...19
Controlling the spacing between the aptamer and the start codon...21

Results and Discussion...23

In vivo screening for switches...23
Controlling the spacing between the aptamer and the start codon...26

Conclusion and Future Directions...30
References...33


List of Figures and Tables
Figure 1. Small molecule ligands found in nature...3
Figure 2. Schematic representation of a riboswitch...4
Figure 3. The catabolic pathway of atrazine...5
Figure 4. Schematic of the SELEX process...6
Figure 5. Summary of the SELEX experiments for atrazine...7
Figure 6. Secondary structure of the atrazine "on" switch at the translational level...8
Figure 7. Randomization of the expression platform...10
Figure 8. The functionality of cheZ...11
Figure 9. The addition of ONPG in beta-galactosidase assays...12
Figure 10. Original atrazine riboswitch sequence...13
Figure 11. Scheme of three-part PCR reactions...15
Figure 12. Primers used in the construction of the N12 and N10 randomized constructs...16
Figure 13. Selection of riboswitches...18
Figure 14. Primers specific to the constructs to decrease the length of the expression platform...21
Figure 15. Results of the N10 randomization motility assay...25
Figure 16. Lynch-Gallivan plot for deletion experiments...27
Figure 17. Secondary structure of the aptamer in the absence and presence of atrazine...28
Figure 18. Creation of an "off" switch from the original atrazine riboswitch...31
Table 1. Activation Ratios of Potential Switches...23

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