Improving a Synthetic Riboswitch that Responds to an Herbicide Público
Longo, Julie Michelle (2011)
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
About this Honors Thesis
School | |
---|---|
Department | |
Degree | |
Submission | |
Language |
|
Research Field | |
Palabra Clave | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
Improving a Synthetic Riboswitch that Responds to an Herbicide () | 2018-08-28 15:15:59 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|