Overview

Abstract

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.

Two-level sRNA regulation. Blue is any target mRNA, red is sRNA and green is 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 promoter activities using synthetic promoter libraries.

Experiment: Testing sRNA

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

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. Read more...

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.

Experiment: Improving araBAD

The dynamic range of the araBAD promoter was expanded. Read more..

Results

The bioinformatic study revealed some interesting constrains for engineering novel sRNAs with our gene silencing tool; a terminal poly-U tail, a putative Hfq binding site, a stemloop whitout sequence constrains, and a terminal stemloop with high sequence conservation. See the results here.

Testing sRNA... See the results here.

improving araBAD... See the results here.

The modeling provides a guideline for determining parameters and design of dynamic range of our gene silencing tool. A simulation revealed fast dynamics of gene silencing. See the results here.