Thesis

Validation of Rnase J1 Function in Escherichia coli Cell System

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Since its discovery 35 years ago, the functional significance of catalytic RNA molecules, called ribozymes, has been better manifested through the analysis of their crystal structures. Hammerhead ribozyme of a plant satellite viroid catalyses a site-specific self-cleavage reaction, driven by an acid-base catalysis, producing RNA products with 2’3’-cyclic phosphate group and 5’-hydroxyl group. In this study, hammerhead ribozyme is utilised to validate the action of RNase J1, a ribonuclease that degrades the 5’-hydroxyl transcripts involved in glmS gene regulation during cell wall biosynthesis in Bacillus subtilis. An in vivo Hammerhead ribozyme system and an inducible-RNase J1 expression is established in BL21(DE3) cells using a two-stage Lambda Red recombineering. The first stage permitted the integration of ammerhead ribozyme fused with reporter EGFP-hDHFR gene at arsB locus of BL21(DE3) genome while the second stage integrated RNase J1 gene behind an Arabinose-inducible P ARA promoter at lacZ locus. Fluorescence assay was performed to evaluate the fluorescence (EGFP-hDHFR) production in the established strains upon graded expression of RNAse J1 under different arabinose concentrations. 49% and 60% reduction of fluorescence intensity were apparent in strain with active Hammerhead ribozyme treated with 0.0012% (w/v) and 0.0037% (w/v) arabinose, respectively, implying RNase J1 degradation of the 5’-hydroxyl tagged EGFP-hDHFR mRNA produced through the ribozyme cleavage. The implication of RNase J1 activity in E. coli in this study upholds the concept of constructing a cell based screening assay for the identification of compounds that target the glmS ribozyme of B. subtilis.

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Series Title

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Call Number

BM 19-030

Publisher

Jakarta : i3L., 2019

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Language

English

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NONE

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