Encapsulation of Autoinducer Sensing Reporter Bacteria in Reinforced Alginate-Based Microbeads

Abstract

Quorum sensing, in which bacteria communities use signaling molecules for inter- and intracellular communication, has been intensively studied in recent decades. In order to fabricate highly sensitive easy-to-handle point of care biosensors that detect quorum sensing molecules, we have developed, as is reported here, reporter bacteria loaded alginate–methacrylate (alginate-MA) hydrogel beads. The alginate-MA beads, which were obtained by electrostatic extrusion, were reinforced by photo-cross-linking to increase stability and thereby to reduce bacteria leaching. In these beads the genetically engineered fluorescent reporter bacterium Escherichia coli pTetR-LasR-pLuxR-GFP (E. coli pLuxR-GFP) was encapsulated, which responds to the autoinducer N-(3-oxododecanoyl)homoserine lactone secreted by Pseudomonas aeruginosa. After encapsulation in alginate-MA hydrogel beads with diameters in the range of 100–300 μm that were produced by an electrostatic extrusion method and rapid photo-cross-linking, the E. coli pLuxR-GFP were found to possess a high degree of viability and sensing activity. The encapsulated bacteria could proliferate inside the hydrogel beads, when exposed to bacteria culture medium. In media containing the autoinducer N-(3-oxododecanoyl)homoserine lactone, the encapsulated reporter bacteria responded with a strong fluorescence signal due to an increased green fluorescent protein (GFP) expression. A prototype dipstick type sensor developed here underlines the potential of encapsulation of viable and functional reporter bacteria inside reinforced alginate–methacrylate hydrogel beads for whole cell sensors for bacteria detection.

Tough Hydrogel-Based Biocontainment of Engineered Organisms for Continuous, Self-Powered Sensing and Computation

“…Alginate forms hydrogels in the presence of di-cationic solutions (e.g., Ca 2+ , Ba 2+ ) and has been used in various biomedical applications [26][27][28][29][30] because of its low cost, negligible cytotoxicity, and mild gelation conditions. However, weak mechanical properties and susceptibility to multiple chemical conditions (such as low pH, citrate, and phosphate) make alginate, as well as other traditional hydrogels, poor solutions for robust physical containment when used on their own [31][32][33] .…”

Optoregulated Protein Release from an Engineered Living Material

“…Alternatively, bacterial strains with different functions can be incorporated in a material. From the materials perspective, various different types of encapsulating matrices such as polyacrylamide, 32 alginate, 33 PEG diacrylate, 34 pluronic, 16 etc. can be achieved and processed into various forms such as microbeads, 33,34 bioprinted scaffolds 16 and even electrospun fibers.…”

Hydrogel Microparticles Functionalized with Engineered Escherichia coli as Living Lactam Biosensors

“…Digital formats of hydrogels, such as microparticles, can be synthesized by microfabrication means, enabling multiplexed sensing in biological samples. Encapsulating whole-cell biosensors in hydrogels have been reported by several groups [30,31]. Futra and coworkers encapsulated Aliivibrio fischeri in alginate microgels for monitoring heavy metal levels in environmental samples [31].…”

“…Futra and coworkers encapsulated Aliivibrio fischeri in alginate microgels for monitoring heavy metal levels in environmental samples [31]. Li and coworkers demonstrated encapsulation of reporter bacteria in alginate microbeads to sense homoserine lactone [30]. In these works, however, the use of hydrogel encapsulation is solely for immobilization and the ease of handling.…”