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Title: De novo engineering of trans-activating riboswitches in E. coli
Author: Prakash, Satya
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2018
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RNA molecules play a major role in cellular processes such as replication, transcription, and translation. As a result, RNA-based engineering methods have emerged as important tools in biotechnology. However, the structure and function of RNA depends on global interactions, which often prevents the use of a modular design strategy, particularly with allosteric conformations. Using RNA secondary structure prediction tools, computational methods can successfully design RNA switches that work in E. coli. The overarching aim of my research is to develop synthetic RNA switches that could be used for regulation and sensing of molecules in living cells. We have engineered RNA based synthetic signal transduction cascade consisting of a single RNA molecule (regazyme, an RNA chimera of an aptazyme with a riboregulator) that upon sensing a ligand (theophylline) self cleaves and releases a riboregulating small RNA. This small RNA binds to a cis-repressed mRNA allowing translation of a reporter protein. This system can be adapted to be induced by other ligands and can be used as a biosensor. I have also integrated a riboregulated RNA switch into the E. coli genome to study its behaviour at single-cell level. This reduces the transcriptional and translational noise in data collection to inform more accurate computational design of RNA regulatory units. We used computational design to engineer higher-order RNA-triggered riboregulators organized as a hierarchical toehold activation cascade. This has been studied in a single cell as well as in a population of E. coli cells. These RNA riboregulators can be used for construction of new, complex and portable synthetic gene circuits. In addition, I have engineered sense and antisense riboregulators consisting of the small RNA reverse complement of a known riboregulator. This riboregulator can transcribe two small RNAs from the same DNA template depending on the direction of transcription. These two small RNAs independently trans-activate translation of their cognate target genes and both RNAs also silence each other by antisense interaction. We have also engineered an RNA-based tunable antiterminator, a TNA-derived adaptor that acts as a signal converter in a genetic circuit, converting a translation signal to a transcription signal (unpublished). I have engineered a minimum alphabet riboregulator that has only three nucleotides (GCU) that currently validating (unpublished). In order to explore the use of directed evolution for the engineering of RNA switches, I am developing an evolution-based system for generation and selection of new biomolecules. These evolved new biomolecules could be used in future medical applications such as molecular sensing. I have been using T7 and P2 bacteriophages as the basis for this evolutionary system. I have engineered the genome of the T7 phage with (regazyme, Riboswitch and riboregulators) using homologous recombination with marker-based selection. These engineered phages can be used to evolve new biomolecules such as other regazymes, riboswitches and riboregulators.
Supervisor: Not available Sponsor: European Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH426 Genetics ; QR Microbiology