Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

<p>Abstract</p> <p>Background</p> <p>A large amount of recombinant proteins can be synthesized in a few hours with <it>Escherichia coli </it>cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of <it>in vitro </it>informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source), and mRNA inactivation and protein degradation (the sink).</p> <p>Results</p> <p>Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an <it>E. coli </it>cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The <it>E. coli </it>mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the <it>Firefly luciferase </it>(Luc) and of the enhanced green fluorescent protein (eGFP). Peptide tags specific to the endogenous <it>E. coli </it>AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured.</p> <p>Conclusions</p> <p>The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in new research areas such as <it>in vitro </it>synthetic biology and systems biology where engineering informational processes requires a quantitative control of mRNA inactivation and protein degradation.</p

Biomolecular resource utilization in elementary cell-free gene circuits

We present a detailed dynamical model of the behavior of transcription-translation circuits in vitro that makes explicit the roles played by essential molecular resources. A set of simple two-gene test circuits operating in a cell-free biochemical 'breadboard' validate this model and highlight the consequences of limited resource availability. In particular, we are able to confirm the existence of biomolecular 'crosstalk' and isolate its individual sources. The implications of crosstalk for biomolecular circuit design and function are discussed

Boron isotope fractionation in soils at Shale Hills CZO

Isotope fractionation of many elements can fingerprint the biogeochemical, weathering and erosion processes that govern the evolution of the Critical Zone (CZ). This study investigates boron isotope fractionation in two soil profiles developed on the same shale bedrock at Shale Hills Critical Zone Observatory. The first soil profile, located at the valley floor, is isotopically similar to the bedrock and appears to have lost boron mostly through the loss of fine particles matter (clays) with no isotopic fractionation. The second soil profile, located at the ridge top appears to be more depleted in boron concentration and isotopically fractionated toward lower values, as expected from mineral dissolution followed by adsorption/co-precipitation processes

Boron isotope fractionation in soils at Shale Hills CZO

Isotope fractionation of many elements can fingerprint the biogeochemical, weathering and erosion processes that govern the evolution of the Critical Zone (CZ). This study investigates boron isotope fractionation in two soil profiles developed on the same shale bedrock at Shale Hills Critical Zone Observatory. The first soil profile, located at the valley floor, is isotopically similar to the bedrock and appears to have lost boron mostly through the loss of fine particles matter (clays) with no isotopic fractionation. The second soil profile, located at the ridge top appears to be more depleted in boron concentration and isotopically fractionated toward lower values, as expected from mineral dissolution followed by adsorption/co-precipitation processes

Symmetry-Breaking Motility

Locomotion of bacteria by actin polymerization, and in vitro motion of spherical beads coated with a protein catalyzing polymerization, are examples of active motility. Starting from a simple model of forces locally normal to the surface of a bead, we construct a phenomenological equation for its motion. The singularities at a continuous transition between moving and stationary beads are shown to be related to the symmetries of its shape. Universal features of the phase behavior are calculated analytically and confirmed by simulations. Fluctuations in velocity are shown to be generically non-Maxwellian and correlated to the shape of the bead.Comment: 4 pages, 2 figures, REVTeX; formatting of references correcte

Soft Listeria: actin-based propulsion of liquid drops

We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pear-like shape under the action of the elastic stresses exerted by the actin comet. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.Comment: 5 pages, 4 figure