Quantifying translational coupling in E. coli synthetic operons using RBS modulation and fluorescent reporters.

Translational coupling is the interdependence of translation efficiency of neighboring genes encoded within an operon. The degree of coupling may be quantified by measuring how the translation rate of a gene is modulated by the translation rate of its upstream gene. Translational coupling was observed in prokaryotic operons several decades ago, but the quantitative range of modulation translational coupling leads to and the factors governing this modulation were only partially characterized. In this study, we systematically quantify and characterize translational coupling in E. coli synthetic operons using a library of plasmids carrying fluorescent reporter genes that are controlled by a set of different ribosome binding site (RBS) sequences. The downstream gene expression level is found to be enhanced by the upstream gene expression via translational coupling with the enhancement level varying from almost no coupling to over 10-fold depending on the upstream gene's sequence. Additionally, we find that the level of translational coupling in our system is similar between the second and third locations in the operon. The coupling depends on the distance between the stop codon of the upstream gene and the start codon of the downstream gene. This study is the first to systematically and quantitatively characterize translational coupling in a synthetic E. coli operon. Our analysis will be useful in accurate manipulation of gene expression in synthetic biology and serves as a step toward understanding the mechanisms involved in translational expression modulation.

Spanning high-dimensional expression space using ribosome-binding site combinatorics.

Protein levels are a dominant factor shaping natural and synthetic biological systems. Although proper functioning of metabolic pathways relies on precise control of enzyme levels, the experimental ability to balance the levels of many genes in parallel is a major outstanding challenge. Here, we introduce a rapid and modular method to span the expression space of several proteins in parallel. By combinatorially pairing genes with a compact set of ribosome-binding sites, we modulate protein abundance by several orders of magnitude. We demonstrate our strategy by using a synthetic operon containing fluorescent proteins to span a 3D color space. Using the same approach, we modulate a recombinant carotenoid biosynthesis pathway in Escherichia coli to reveal a diversity of phenotypes, each characterized by a distinct carotenoid accumulation profile. In a single combinatorial assembly, we achieve a yield of the industrially valuable compound astaxanthin 4-fold higher than previously reported. The methodology presented here provides an efficient tool for exploring a high-dimensional expression space to locate desirable phenotypes.

Primary care physicians' use of an electronic medical record system: a cognitive task analysis.

OBJECTIVE: To describe physicians' patterns of using an Electronic Medical Record (EMR) system; to reveal the underlying cognitive elements involved in EMR use, possible resulting errors, and influences on patient-doctor communication; to gain insight into the role of expertise in incorporating EMRs into clinical practice in general and communicative behavior in particular. DESIGN: Cognitive task analysis using semi-structured interviews and field observations. PARTICIPANTS: Twenty-five primary care physicians from the northern district of the largest health maintenance organization (HMO) in Israel. RESULTS: The comprehensiveness, organization, and readability of data in the EMR system reduced physicians' need to recall information from memory and the difficulty of reading handwriting. Physicians perceived EMR use as reducing the cognitive load associated with clinical tasks. Automaticity of EMR use contributed to efficiency, but sometimes resulted in errors, such as the selection of incorrect medication or the input of data into the wrong patient's chart. EMR use interfered with patient-doctor communication. The main strategy for overcoming this problem involved separating EMR use from time spent communicating with patients. Computer mastery and enhanced physicians' communication skills also helped. CONCLUSIONS: There is a fine balance between the benefits and risks of EMR use. Automaticity, especially in combination with interruptions, emerged as the main cognitive factor contributing to errors. EMR use had a negative influence on communication, a problem that can be partially addressed by improving the spatial organization of physicians' offices and by enhancing physicians' computer and communication skills.