On a Robust, Sensitive Cell-Free Method for Pseudomonas Sensing and Quantification in Microfluidic Templated Hydrogels

Abstract

Through the use of droplet microfluidics to integrate cell-free activity into inert hydrogel beads, we have developed a platform that can perform biologically relevant functions without the need for cells. Specifically, cell-free lysates serve a utility in performing cellular functions and providing biologically relevant metabolic products without requiring the optimal biological conditions for cell growth and proliferation. By teasing out specific biological components that enable transcription and translation to occur, these cell-like functions can be reconstituted in vitro without requiring the entire cell and milieu of cellular organelles. This enables the optimization of synthetic biological circuits, either by concentration or logic switches, simply through the addition or removal of genetic components (plasmids, inducers, or repressors) of regulatory elements. Here, we demonstrate an application of cell-free processes that is robust and portable, independent of a substrate, to apply for sensing and reporting functions of a quorum-sensing molecule N-3-oxododecanoyl homoserine lactone (3OC12HSL) found crucial for pathological Pseudomonas aeruginosa infection. We develop an agarose bead platform that is easily adaptable and simply programmable to fit a variety of biological and chemical sensing applications for the utility of ease of delivery and activation in remote environments—even in conditions with very little hydration.

Effective Use of Linear DNA in Cell-Free Expression Systems

“…LETs have also been spotted on glass slides and aligned to microfluidic devices enclosing each LET into a reaction chamber ( Gerber et al, 2009 ). Microfluidic devices have also been used to encapsulate LET-containing CFEs (with GamS inhibitor) into agarose hydrogel beads, which then withstood lyophilization, storage, and rehydration into a functional sensor for P. aeruginosa quorum-sensing molecules ( Seto 2019 ). Moreover, with sufficient protection, LETs can be used in continuous reaction conditions as well.…”

Editorial for the Special Issue of 10th Anniversary of Micromachines

“…A total of 52 papers, including 42 original research articles, 9 review articles, and 1 brief report, are published in this Special Issue. A total of 14 out of the 52 papers in the Special Issue are selected as feature papers [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ].…”

“…A subtractive glass 3D printing technique was reported by Peng Wang et al [ 10 ]. The authors demonstrated the fabrication of macro-scale 3D glass objects of large heights up to ~3.8 cm with an identical lateral and longitudinal feature size of ~20 μm.…”