Overview

Abstract

Two-level sRNA regulation. Blue is any target mRNA, red is sRNA and green is trap-RNA.

Small regulatory RNA is an active area of research with untapped possibilities for application in biotechnology. Such applications include convenient gene silencing and fine-tuning of gene expression, which are currently cumbersome processes restricted to well studied bacteria. We have investigated a novel type of RNA regulation based on the chitobiose system, where the inhibition caused by a small RNA is relieved by another small RNA called trap-RNA. We explore the possibility of using the system to uniquely target and repress any gene of interest, potentially providing unprecedented specificity and control of gene silencing. We furthermore constructed araBAD promoters with varying dynamic ranges using synthetic promoter libraries.

Bioinformatics

A bioinformatics study was performed to investigate the possibilities of engineering the trap-RNA system to target any gene. The study elucidates interesting features of sequence and secondary structure conservation guiding future application.

Experiments 1

Verifying that the envisioned small RNA based gene silencing is possible. Plasmids containing and strains deleted for the components were constructed providing a biological model.

Experiments 2

The dynamic range of the araBAD promoter was expanded.

Modeling

A framework for characterization was developed to guide rational design and test hypotheses. Steady state analysis revealed that each trap-RNA system has a characteristic fold repression.