Characterizing the Structure-Function Relationship of a Naturally Occurring RNA Thermometer.

A large number of bacteria have been found to govern virulence and heat shock responses using temperature-sensing RNAs known as RNA thermometers. A prime example is the agsA thermometer known to regulate the production of the AgsA heat shock protein in Salmonella enterica using a "fourU" structural motif. Using the SHAPE-Seq RNA structure-probing method in vivo and in vitro, we found that the regulator functions by a subtle shift in equilibrium RNA structure populations that leads to a partial melting of the helix containing the ribosome binding site.

Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions.

The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media.

Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems.

The centrality of RNA for engineering gene expression.

Synthetic biology holds promise as both a framework for rationally engineering biological systems and a way to revolutionize how we fundamentally understand them. Essential to realizing this promise is the development of strategies and tools to reliably and predictably control and characterize sophisticated patterns of gene expression. Here we review the role that RNA can play towards this goal and make a case for why this versatile, designable, and increasingly characterizable molecule is one of the most powerful substrates for engineering gene expression at our disposal.

Engineering alternate cooperative-communications in the lactose repressor protein scaffold.

To expand our understanding of the hallmarks of allosteric control we used directed-evolution to engineer alternate cooperative communication in the lactose repressor protein (LacI) scaffold. Starting with an I(s) type LacI mutant D88A (i.e.