Isolated Reporter Bacteria in Supramolecular Hydrogel Microwell Arrays

Abstract

The combination of supramolecular hydrogels formed by low molecular weight gelator self-assembly via noncovalent interactions within a scaffold derived from polyethylene glycol (PEG) affords an interesting approach to immobilize fully functional, isolated reporter bacteria in novel microwell arrays. The PEG-based scaffold serves as a stabilizing element and provides physical support for the self-assembly of the C2-phenyl-derived gelator on the micrometer scale. Supramolecular hydrogel microwell arrays with various shapes and sizes were used to isolate single or small numbers of Escherichia coli TOP10 pTetR-LasR-pLuxR-GFP. In the presence of the autoinducer N-(3-oxododecanoyl) homoserine lactone, the entrapped E. coli in the hydrogel microwell arrays showed an increased GFP expression. The shape and size of microwell arrays did not influence the fluorescence intensity and the projected size of the bacteria markedly, while the population density of seeded bacteria affected the number of bacteria expressing GFP per well. The hydrogel microwell arrays can be further used to investigate quorum sensing, reflecting communication in inter- and intraspecies bacterial communities for biology applications in the field of biosensors. In the future, these self-assembled hydrogel microwell arrays can also be used as a substrate to detect bacteria via secreted autoinducers.

Autoinducer Sensing Microarrays by Reporter Bacteria Encapsulated in Hybrid Supramolecular‐Polysaccharide Hydrogels

“…The fluorescence intensity, normalized to the cell density (fluorescence intensity divided by OD 600 ) and further divided by that observed in the control culture (without exposition to 3OC 12 HSL), was used as a relative measure for GFP expression. In the presence of 1 × 10 −6 to 1 × 10 −8 mol L −1 3OC 12 HSL, the normalized fluorescence intensity was ≈12 times higher than in the absence of autoinducers, which is identical to the results of bacteria entrapped in PDB gels . For bacteria in both LB medium and in the bulk hydrogels, no significant difference in relative GFP expression was observed for all the 3OC 12 HSL concentrations tested.…”

“…The estimated bacteria numbers per well were (2.9 ± 0.3) × 10 4 . This large number of bacteria per well could average out any possible variance caused by single bacteria …”

“…In the presence of 1 × 10 −6 to 1 × 10 −8 mol L −1 3OC 12 HSL, the normalized fluorescence intensity was ≈12 times higher than in the absence of autoinducers, which is identical to the results of bacteria entrapped in PDB gels. [32] For bacteria in both LB medium and in the bulk hydrogels, no significant difference in relative GFP expression was observed for all the 3OC 12 HSL concentrations tested. For the following experiments with biosensor arrays, 1 × 10 −6 mol L −1 3OC 12 HSL was used.…”

“…E. coli TOP10 pTetR‐LasR‐pLuxR‐GFP ( E. coli pLuxR‐GFP as abbreviation) was cultured according to published protocols …”

“…[29] The controllable physical and biochemical properties from rational molecular design are used to tune the behavior and functionality of the enclosed cells. [30] While we showed the feasibility of supramolecular hydrogels formed by low molar mass gelator self-assembly for reporter bacteria encapsulation in a microarray format with single bacteria per well for N-acyl homoserine lactone autoinducer-reporting Escherichia coli pTetR-LasR-pLuxR-green fluorescent protein (GFP) (E. coli pLuxR-GFP), [31,32] the utilized supramolecular hydrogels possessed poor mechanical properties.…”