Phenotypic Patterning through Copy Number Adaptation to Environmental Gradients.

We recently described a paradigm for engineering bacterial adaptation using plasmids coupled to the same origin of replication. In this study, we use plasmid coupling to generate spatially separated and phenotypically distinct populations in response to heterogeneous environments. Using a custom microfluidic device, we continuously tracked engineered populations along induced gradients, enabling an in-depth analysis of the spatiotemporal dynamics of plasmid coupling. Our observations reveal a pronounced phenotypic separation within 4 h exposure to an opposing gradient of AHL and arabinose. Additionally, by modulating the burden strength balance between coupled plasmids, we demonstrate the inherent limitations and tunability of this system. Intriguingly, phenotypic separation persists for an extended time, hinting at a biophysical spatial retention mechanism reminiscent of natural speciation processes. Complementing our experimental data, mathematical models provide invaluable insights into the underlying mechanisms and guide optimization of plasmid coupling for prospective applications of environmental copy number adaptation engineering across separated domains.

Point-of-Care Analyte Quantification and Digital Readout via Lysate-Based Cell-Free Biosensors Interfaced with Personal Glucose Monitors.

Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate the production of the reporter enzyme ß-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase the end point signal output. However, this lysate metabolism did allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, our results show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.

Point-of-care biomarker quantification enabled by sample-specific calibration.

Easy-to-perform, relatively inexpensive blood diagnostics have transformed at-home healthcare for some patients, but they require analytical equipment and are not easily adapted to measuring other biomarkers. The requirement for reliable quantification in complex sample types (such as blood) has been a critical roadblock in developing and deploying inexpensive, minimal-equipment diagnostics. Here, we developed a platform for inexpensive, easy-to-use diagnostics that uses cell-free expression to generate colored readouts that are visible to the naked eye, yet quantitative and robust to the interference effects seen in complex samples. We achieved this via a parallelized calibration scheme that uses the patient sample to generate custom reference curves. We used this approach to quantify a clinically relevant micronutrient and to quantify nucleic acids, demonstrating a generalizable platform for low-cost quantitative diagnostics.