Tools and Principles for Microbial Gene Circuit Engineering

Bradley, Robert W.; Buck, Martin; Wang, Baojun

doi:10.1016/j.jmb.2015.10.004

Cited by 88 publications

(82 citation statements)

References 227 publications

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“…Strict control of transcription can reduce the metabolic burden of production and improve the processes’ stability (Weisse et al, ). Genetic sensors and circuits can provide stringent pathway control by gene regulation (Bradley et al, ; O'Connor and Adams, ), improving the products’ yields and titers (Chubukov et al, ; Du et al, ; Lo et al, ). As biological processes transition from lab scale to industrial scale production, biological sensors are needed to link control of transcription to specific and relevant signals (Hoynes‐O'Connor and Moon, ).…”

Section: Introductionmentioning

confidence: 99%

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Immethun

DeLorenzo

Focht

et al. 2017

Biotech & Bioengineering

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Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Immethun

DeLorenzo

Focht

et al. 2017

Biotech & Bioengineering

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“…[1] This development has led to the introduction of new biological functions into organisms such as the capability to produce highly complex organic molecules, like pharmaceuticals, food additives, and fuels. [1] This development has led to the introduction of new biological functions into organisms such as the capability to produce highly complex organic molecules, like pharmaceuticals, food additives, and fuels.…”

Section: Introductionmentioning

confidence: 99%

Continuous Production of a Shelf‐Stable Living Material as a Biosensor Platform

Schulz‐Schönhagen

Lobsiger

Stark

2019

Adv Materials Technologies

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analyze biomarkers in human diseases, such as nitrogen oxides involved in inflammation response. [11] This organismbased type of sensor systems is referred to as whole-cell biosensors, since the sensing functionality and signal transduction require intact, living cells. The basic working principle of a whole-cell biosensor relies on an input, which activates a certain pathway in the cell leading to an output signal such as color, fluorescence, or luminescence that can be measured by the user. [12,13] Cells genetically engineered to recognize a specific target can be produced at low cost compared to other biological diagnostic systems such as enzyme-or antibody-based assays. Nevertheless, the readout of whole-cell biosensors and the quantification of the measured signal require high-priced laboratory equipment such as fluorometers, spectrophotometers, or luminometers and trained personnel to operate these devices.An alternative approach for the quantification of whole-cell biosensors systems has been presented by Mora et al. by embedding genetically modified Escherichia coli cells into an agarose hydrogel. [14] The concentration of different sugars in solution, which were applied to the surface of the hydrogel, was quantified by the direct correlation between the diffusion in the material and the fluorescence induction by the same sugars in the E. coli cells. This living material-based analytical sensor showed quantification precision comparable to those of HPLC-MS and a fluorometric lactose/galactose enzymatic assay. As a drawback, the system requires a cold chain to maintain the viability of the embedded organisms, resulting in limited shelf life of the sensor. Additionally, the material was produced in a batch process and had, due to the fragile nature of the agarose gel, limited mechanical stability. Both factors limit the production to a laboratory scale and the use to a controlled environment, where continuous cooling and short periods of time between production and application are ensured. These restrictions forbid the application of the sensors in areas where an undeveloped infrastructure would compromise the functionality of the material.To facilitate the use of whole-cell biosensors in the field, a platform is required that is versatile in its application. Additionally, it has to be cheap in production, robust toward storage and changing environmental conditions, and can be The advancements made in biological engineering have led to the development of whole-cell biosensors and their successful application in diagnostics. However, due to the sensitivity of the systems, as well as the need for advanced readout devices, the use of such sensors is limited to a laboratory environment. Here, a biosensor platform for the diffusion-based quantification of a small molecule analyte is presented. The platform consists of Bacillus subtilis endospores carrying a genetic reporter construct. The spores are embedded in a poly(vinyl alcohol) matrix casted to a poly(ethylene terephthalate) support material. It is shown th...

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“…RNA is an attractive substrate for this, due to its substantial regulatory and sensing potential [2,3] as well as the availability of computational tools to estimate its behaviour [1,13]. Indeed, RNA molecules can be designed to sense extremely specific signals such as metabolites or other RNAs [7] as well as more global signals such as temperature [8].…”

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confidence: 99%

Design of a Toolbox of RNA Thermometers

et al. 2017

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Abstract. Biomolecular temperature sensors can be used for efficient control of large-volume bioreactors, for spatiotemporal control and imaging of gene expression, as well as to engineer robustness to temperature in biomolecular circuit design. While RNA-based sensors, called 'thermometers', have been investigated in natural and synthetic contexts, an important challenge is to design different responses to temperature, differing in sensitivities and thresholds. We address this issue using experimental measurements in cells and in cell-free biomolecular 'breadboards' in combination with computations of RNA thermodynamics. We designed a library of RNA thermometers, finding, com-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017269 doi: bioRxiv preprint first posted online Mar. 30, 2015; putationally, that it could contain a multiplicity of responses to temperature. We constructed this library and found a wide range of responses to temperature, ranging from 3.5-fold to over 10-fold in the temperature range 29 • C -37 • C. These were largely linear responses with over 10-fold difference in slopes. We correlated the measured responses with computational expectations, finding that while there was no strong correlation in the individual values, the overall trends were similar. These results present a toolbox of RNA-based circuit elements with varying temperature sensitivities.

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Tools and Principles for Microbial Gene Circuit Engineering

Cited by 88 publications

References 227 publications

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Continuous Production of a Shelf‐Stable Living Material as a Biosensor Platform

Design of a Toolbox of RNA Thermometers

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