Eric A. Davidson

Engineering Escherichia coli to see light

We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to ‘print’ complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.

Toward automated nucleic acid enzyme selection

Abstract Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5 iodine residue and the ligation substrate contained a 3 phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing beadbound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic observation of the selections as they progress.

An in vitro Autogene

Recent technological advances have allowed development of increasingly complex systems for in vitro evolution. Here, we describe an in vitro autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture. Compartmentalization of individual templates within a water-in-oil emulsion links genotype and phenotype, allowing evolution.

Directed Evolution of Proteins In Vitro Using Compartmentalization in Emulsions

This unit describes a protocol for the directed evolution of proteins utilizing in vitro compartmentalization. This method uses a large number of independent in vitro transcription and translation (IVTT) reactions in water droplets suspended in an oil emulsion to enable selection of proteins that bind a target molecule. Protein variants that bind the target also bind to and allow recovery of the genes that encoded them. This protocol serves as a basis for carrying out selections in emulsions, and can potentially be modified to select for other functionalities, including catalysis. This selection method is advantageous compared to alternative selection protocols due to the ability to screen through very large‐size libraries and the ability to express and screen or select for functions that would otherwise be toxic or inaccessible to in vivo selections and screens. Curr. Protoc. Mol. Biol. 87:24.6.1‐24.6.12.

Developing RNA tools for engineered regulatory systems

RNA has been shown recently to play a prominent role in regulating gene expression in a wide variety of organisms. Natural examples of RNA-dependent gene regulation include modulation of transcription (Wassarman and Storz, 2000; Winkler et al., 2002), translation (Moller-Jensen eral., 2001; Moller et al., 2002; Bartel, 2004), and the temporal stability of mRNA (Masse et al., 2003; Meister and Tuschl, 2004; Winkler et al., 2004). Environmental perturbations, such as temperature (Hoe and Goguen, 1993; Johansson et al., 2002), osmolarity (Andersen et al., 1987; Chen et al., 2004), oxidative stress (Altuvia et al., 1997), and chemistry (such as the presence of metabolites or toxins, reviewed in Mandal and Breaker, 2004) can all trigger RNA-mediated changes in gene expression. In parallel, researchers have begun to use engineered RNAs as regulatory molecules for artificial genetic circuits. The same mechanisms (base-pairing and ligand-induced conformational change) that make RNA a ’natural’ choice for gene regulation are also amenable to rational engineering or directed evolution efforts. Nonetheless, the clever machines that nature has crafted have accelerated the use of artificial regulatory RNAs. Therefore, we will initially consider the natural RNA tools and mechanisms that are available to researchers, then examine how these tools are being applied in artificial genetic circuits. Ultimately, though, both the adaptation of natural, and the de nova development of artificial, RNA-based regulatory mechanisms should foment the production of much more complex artificial genetic circuits, potentially including ’smart’ nucleic acid drugs that regulate their own production and release.