Biology subjects

Thavarajah, W.

Publications and source records attributed to Thavarajah, W..

3 recordsLinked to original sources

bioRxiv2024

Semi-automated Production of Cell-free Biosensors

Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including being able to be assembled, freeze-dried, distributed, and then used at the point-of-need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semi-automated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride sensing cell-free biosensors and demonstrate that they perform closely to expected detection outcomes.

synthetic biology↗

bioRxiv2022

Robust and tunable performance of a cell-free biosensor encapsulated in lipid vesicles

Cell-free systems have enabled the development of genetically encoded biosensors to detect a range of environmental and biological targets. By encapsulating these systems in synthetic membranes, we can reintroduce features of the cell membrane, including molecular containment and selective permeability, which together could significantly enhance sensing capabilities. Here, we demonstrate robust and tunable performance of an encapsulated fluoride riboswitch inside of lipid vesicles. The riboswitch remains active upon encapsulation in lipid vesicles and responds to fluoride added to the surrounding solution. We find the sensitivity of the encapsulated sensor can be tuned by varying membrane composition. We then show that encapsulation protects the sensor from degradation by the sample and use two types of genetically encoded outputs to detect fluoride in real-world samples. This work establishes the feasibility of vesicle-encapsulated cell-free systems to detect environmentally relevant small molecules.

synthetic biology↗

bioRxiv2019

Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 parts per million, the EPAs most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-of-principle for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

synthetic biology↗