Biology subjects

Shahein, A.

Publications and source records attributed to Shahein, A..

2 recordsLinked to original sources

bioRxiv2023

A workcell 1.0 for programmable and controlled operation of multiple fluidic chips in parallel.

We developed a versatile lab-on-chip (LOC) workcell that enables the design and automatic execution of experiments on LOC devices, improving how we establish, optimize, and productionalize LOC processes. Key features include direct docking and cooling of native laboratory tubes, programmable reagent mixing and dilutions, parallel operation of multiple chips, precise flowrate and pressure control, clogging detection and response, programmable microscope control, chip temperature regulation, and scheduled cleaning. All functionality is controlled seamlessly from an easy-to-write protocol file, and based on extensible hardware and software infrastructures to promote community development. To showcase the platforms use and versatility, we demonstrate a series of 5 different automated experiments at varying levels of complexity, executed across both Quake-valve and droplet microfluidic systems. In particular, the workcell was instructed to map the parameter regime that generates viable droplets, to allow a user to select diameters and production frequencies of interest for single bacterial cell encapsulation. Furthermore, three out of three days in a row, the platform successfully performed a complex 15.5h long experiment, integrating in a single automated protocol the full core workflow required by a typical protein-characterization lab: protein expression, purification, dilution generation, and quantitative binding characterization (generating 55296 images in the process). Experiments conducted through the workcell are easier to set up, offer increased control over experiment conditions and parameters, and can be heavily parallelized.

bioengineering↗

bioRxiv2021

Systematic analysis of low-affinity transcription factor binding site clusters in vitro and in vivo establishes their functional relevance

Transcription factor binding to a single binding site and its functional consequence in a promoter context are beginning to be relatively well understood. However, binding to clusters of sites has yet to be characterized in depth, and the functional relevance of binding site clusters remains uncertain. We employed a high-throughput biochemical method to characterize transcription factor binding to clusters varying across a range of affinities and configurations. We found that transcription factors can bind concurrently to overlapping sites, challenging the notion of binding exclusivity. Furthermore, compared to an individual high-affinity binding site, small clusters with binding sites an order of magnitude lower in affinity give rise to higher mean occupancies at physiologically-relevant transcription factor concentrations in vitro. To assess whether the observed in vitro occupancies translate to transcriptional activation in vivo, we tested low-affinity binding site clusters by inserting them into a synthetic minimal CYC1 and the native PHO5 S. cerevisiae promoter. In the minCYC1 promoter, clusters of low-affinity binding sites can generate transcriptional output comparable to a promoter containing three consensus binding sites. In the PHO5 promoter, replacing the native Pho4 binding sites with clusters of low-affinity binding sites recovered activation of these promoters as well. This systematic characterization demonstrates that clusters of low-affinity binding sites achieve substantial occupancies, and that this occupancy can drive expression in eukaryotic promoters.

biophysics↗