BackgroundStandardized and well-characterized genetic building blocks are a prerequisite for the convenient and reproducible assembly of novel genetic modules and devices. While numerous standardized parts exist for Escherichia coli, such tools are still missing for the Gram-positive model organism Bacillus subtilis. The goal of this study was to develop and thoroughly evaluate such a genetic toolbox.ResultsWe developed five BioBrick-compatible integrative B. subtilis vectors by deleting unnecessary parts and removing forbidden restriction sites to allow cloning in BioBrick (RFC10) standard. Three empty backbone vectors with compatible resistance markers and integration sites were generated, allowing the stable chromosomal integration and combination of up to three different devices in one strain. In addition, two integrative reporter vectors, based on the lacZ and luxABCDE cassettes, were BioBrick-adjusted, to enable β-galactosidase and luciferase reporter assays, respectively. Four constitutive and two inducible promoters were thoroughly characterized by quantitative, time-resolved measurements. Together, these promoters cover a range of more than three orders of magnitude in promoter strength, thereby allowing a fine-tuned adjustment of cellular protein amounts. Finally, the Bacillus BioBrick Box also provides five widely used epitope tags (FLAG, His10, cMyc, HA, StrepII), which can be translationally fused N- or C-terminally to any protein of choice.ConclusionOur genetic toolbox contains three compatible empty integration vectors, two reporter vectors and a set of six promoters, two of them inducible. Furthermore, five different epitope tags offer convenient protein handling and detection. All parts adhere to the BioBrick standard and hence enable standardized work with B. subtilis. We believe that our well-documented and carefully evaluated Bacillus BioBrick Box represents a very useful genetic tool kit, not only for the iGEM competition but any other BioBrick-based project in B. subtilis.
The arabinose utilization system of Escherichia coli displays a stochastic all-or-nothing response at intermediate levels of arabinose, where the population divides into a fraction catabolizing the sugar at a high rate (on-state) and a fraction not utilizing arabinose (off-state). Here we study this decision process in individual cells, focusing on the dynamics of the transition from the off- to the on-state. Using quantitative time-lapse microscopy, we determine the time delay between inducer addition and fluorescence onset of a GFP reporter. Through independent characterization of the GFP maturation process, we can separate the lag time caused by the reporter from the intrinsic activation time of the arabinose system. The resulting distribution of intrinsic time delays scales inversely with the external arabinose concentration, and is compatible with a simple stochastic model for arabinose uptake. Our findings support the idea that the heterogeneous timing of gene induction is causally related to a broad distribution of uptake proteins at the time of sugar addition.
Intraoperative use of thoracic epidural catheter reduced stress response and prevented stress-induced perioperative impairment of proinflammatory lymphocyte function.
Sensing of and responding to environmental changes are of vital importance for microbial cells. Consequently, bacteria have evolved a plethora of signaling systems that usually sense biochemical cues either via direct ligand binding, thereby acting as “concentration sensors,” or by responding to downstream effects on bacterial physiology, such as structural damage to the cell. Here, we describe a novel, alternative signaling mechanism that effectively implements a “flux sensor” to regulate antibiotic resistance. It relies on a sensory complex consisting of a histidine kinase and an ABC transporter, in which the transporter fulfills the dual role of both the sensor of the antibiotic and the mediator of resistance against it. Combining systems biological modeling with in vivo experimentation, we show that these systems in fact respond to changes in activity of individual resistance transporters rather than to changes in the antibiotic concentration. Our model shows that the cell thereby adjusts the rate of de novo transporter synthesis to precisely the level needed for protection. Such a flux-sensing mechanism may serve as a cost-efficient produce-to-demand strategy, controlling a widely conserved class of antibiotic resistance systems.
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