BackgroundPositive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors.ResultsWe developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity.ConclusionsThe advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.
The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc.
Whole-cell biosensors (WCBs) have been designed to detect As(III), but most suffer from poor sensitivity and specificity. In this paper, we developed an arsenic WCB with a positive feedback amplifier in Escherichia coli DH5␣. The output signal from the reporter mCherry was significantly enhanced by the positive feedback amplifier. The sensitivity of the WCB with positive feedback is about 1 order of magnitude higher than that without positive feedback when evaluated using a halfsaturation As(III) concentration. The minimum detection limit for As(III) was reduced by 1 order of magnitude to 0.1 M, lower than the World Health Organization standard for the arsenic level in drinking water, 0.01 mg/liter or 0.13 M. Due to the amplification of the output signal, the WCB was able to give detectable signals within a shorter period, and a fast response is essential for in situ operations. Moreover, the WCB with the positive feedback amplifier showed exceptionally high specificity toward As(III) when compared with other metal ions. Collectively, the designed positive feedback amplifier WCB meets the requirements for As(III) detection with high sensitivity and specificity. This work also demonstrates the importance of genetic circuit engineering in designing WCBs, and the use of genetic positive feedback amplifiers is a good strategy to improve the performance of WCBs. IMPORTANCE Arsenic poisoning is a severe public health issue. Rapid and simple methods for the sensitive and specific monitoring of arsenic concentration in drinking water are needed. In this study, we designed an arsenic WCB with a positive feedback amplifier. It is highly sensitive and able to detect arsenic below the WHO limit level. In addition, it also significantly improves the specificity of the biosensor toward arsenic, giving a signal that is about 10 to 20 times stronger in response to As(III) than to other metals. This work not only provides simple but effective arsenic biosensors but also demonstrates the importance of genetic engineering, particularly the use of positive feedback amplifiers, in designing WCBs.A rsenic (As)-contaminated groundwater, occurring from mining or agriculture or natural contamination due to the abundance of arsenic in the Earth's crust, is a serious global health issue. Long-term exposure to arsenic can result in various diseases including cancers (1, 2). It is estimated that over 100 million people worldwide may be at risk from consuming water contaminated with arsenic (3). The World Health Organization (WHO) has recommended 0.01 mg/liter (0.13 M) as a safe permissible level for arsenic in drinking water (4), and the Food and Agriculture Organization (FAO) has set a maximum contamination level (MCL) for arsenic of 0.01 mg/liter in irrigation water (5).
SummaryAutoregulatory gene circuits can be physically encoded within the genome in a number of different configurations. By physical encoding, we mean the orientation and relative proximity of the genes within the circuit. In this work, we quantified the behaviour of an inducible, negatively autoregulated gene circuit arranged in different transcriptional configurations using the tetRA circuit from Tn10 as our basis. Mathematical modelling predicted that circuits arranged in configurations where the expression of the transcription factor is decoupled from its target genes afforded more flexibility relative to configurations where expression is coupled. We found that these decoupled configurations reduced the concentration of transcription factor needed to regulate inducible expression from the circuit. As lower concentrations of transcription factor were required, these decoupled configurations could also be activated at much lower concentrations of the inducer. We experimentally validated these predictions in Escherichia coli by comparing the response of synthetic circuits based on the tetRA circuit arranged in different configurations. Collectively, these results provide one example of how the arrangement of a gene circuit within the genome can affect its behaviour.
Bacteria employ a modified two-component system for chemotaxis, where the receptors form ternary complexes with CheA histidine kinases and CheW adaptor proteins. These complexes are arranged in semi-ordered arrays clustered predominantly at the cell poles. The prevailing models assume that these arrays are static and reorganize only locally in response to attractant binding. Recent studies have shown, however, that these structures may in fact be much more fluid. We investigated the localization of the chemotaxis signaling arrays in Bacillus subtilis using immunofluorescence and live cell fluorescence microscopy. We found that the receptors were localized in clusters at the poles in most cells. However, when the cells were exposed to attractant, the number exhibiting polar clusters was reduced roughly 2-fold, whereas the number exhibiting lateral clusters distinct from the poles increased significantly. These changes in receptor clustering were reversible as polar localization was reestablished in adapted cells. We also investigated the dynamic localization of CheV, a hybrid protein consisting of an N-terminal CheW-like adaptor domain and a C-terminal response regulator domain that is known to be phosphorylated by CheA, using immunofluorescence. Interestingly, we found that CheV was localized predominantly at lateral clusters in unstimulated cells. However, upon exposure to attractant, CheV was found to be predominantly localized to the cell poles. Moreover, changes in CheV localization are phosphorylation-dependent. Collectively, these results suggest that the chemotaxis signaling arrays in B. subtilis are dynamic structures and that feedback loops involving phosphorylation may regulate the positioning of individual proteins.Many motile bacteria employ for chemotaxis a modified two-component system to sense and respond to chemicals, where the receptors form ternary complexes with the CheA histidine kinase and the CheW adaptor protein (1, 2). The clustering of these ternary complexes into semi-ordered hexagonal lattices has been documented in multiple species (3) and is presumably conserved in all chemotactic bacteria where the three proteins are found. These arrays are thought to amplify the response to attractant binding (4,5). A number of models have specifically proposed that cooperative interactions between the receptors within these arrays enable bacteria to sense small differences in the number of attractant-bound receptors over a wide range of concentrations (see Ref. 6).Multiple studies have investigated the structure and molecular determinants of these clusters (e.g. Refs. 7 and 8) along with their role in signal transduction. In Escherichia coli, the receptors form mixed trimers of receptor homodimers. These trimers are believed to form the basic building blocks for the larger clusters, which range in size from tens to thousands of receptors (9). These clusters are found predominantly at the cell poles, although they are also found along the lateral length of the cell. Attractant binding, which inhibi...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.