Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

Abstract

Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life. Specifically, we have employed spore-forming bacteria such as Bacillus subtilis and Bacillus megaterium for development of luminescent sensing systems for two model analytes, namely, arsenic and zinc. These sensing cells were converted to spores, which can then be "revived" (germinated) at a later time to generate viable and metabolically active cells. Herein, we demonstrate that these spore-based sensing systems retained their analytical performance, in terms of detection limit, dynamic range, and reproducibility, after storage at room temperature for at least 6 and 8 months, respectively, as well as after three cycles where the cells alternated between being dormant or active, i.e., sporulation-germination cycles. The ability to cycle the sensing cells between active and dormant states prolongs the cell's lifetimes and increases their robustness and ruggedness, thus making them more amenable for field applications. In addition, the small size of spores allows for their easy transport and incorporation in miniaturized portable devices. Finally, we envision that this novel strategy could expand the use of whole-cell biosensors for on-site sensing not only in mild environments but also in harsh environments and locations where there is no easy access to a laboratory, e.g., in developing countries.

Sposensor: A Whole-Bacterial Biosensor That Uses Immobilized <i>Bacillus subtilis</i> Spores and a One-Step Incubation/Detection Process

“…These dormant highly resistant cells can easily differentiate into responding cells after a germination step. As a matter of fact, while this work was in progress, the ability of spore-based systems to retain their detection properties was investigated [Date et al, 2007]. The authors showed that spores were effectively able to retain their analytical performance (in terms of detection limit, dynamic range and reproducibility) after either a long storage period or several cycles of germination/sporulation.…”

“…In this regard, colorimetric assay is highly recommended because no special tool is required to detect, at least qualitatively, the color of the product of the reaction. Immobilizing bacteria or the use of spores were proposed as issues regarding robustness problems [Date et al, 2007].…”

Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

“…The natural ability of some microorganisms to form highly resilient spores can also be taken advantage of as a long-term storage solution. Date et al [14] converted spore-forming Bacillus cells into bioreporters with shelf-lives of up to 8 months, and further showed that they could be repeatedly cycled between their active and dormant states with little ancillary effect on their sensing capabilities.…”

Beyond Genetic Engineering: Technical Capabilities in the Application Fields of Biocatalysis and Biosensors

“…A number of reports have described the use of B. subtilis in biosensors. Luciferase-based biosensors have been reported for arsenic, antimony, cadmium and lead (Tauriainen et al 1997(Tauriainen et al , 1998, and β-galactosidase-based chemiluminescent and chromogenic endospore biosensors for arsenic, bacitracin and zinc (Date et al 2007(Date et al , 2010. We have previously reported an endospore-based arsenic biosensor with a chromogenic visual response for easy detection in the field and are currently involved in a collaboration to develop an improved version of this (Arsenic Biosensor Collaboration 2013).…”