Biology subjects

Benzinger, D.

Publications and source records attributed to Benzinger, D..

7 recordsLinked to original sources

bioRxiv2025

Predictable Engineering of Signal-DependentCis-Regulatory Elements

Cis-regulatory elements (CREs) control how genes respond to external signals, but the principles governing their structure and function remain poorly understood. While differential transcription factor binding is known to regulate gene expression, how CREs integrate the amount and combination of inputs to secure precise spatiotemporal profiles of gene expression remains unclear. Here, we developed a high-throughput combinatorial screening strategy, that we term NeMECiS, to investigate signal- dependent synthetic CREs (synCREs) in differentiating mammalian stem cells. By concatenating fragments of functional CREs from genes that respond to Sonic Hedgehog in the developing vertebrate neural tube, we found that CRE activity follows hierarchical design rules. While individual 200-base-pair fragments showed minimal activity, their combinations generated thousands of functional signal-responsive synCREs, many exceeding the activity of natural sequences. Statistical modelling revealed CRE function can be decomposed into specific quantitative contributions in which sequence fragments combine through a multiplicative rule, tuned by their relative positioning and spacing. These findings provide a predictive framework for CRE redesign, which we used to engineer synthetic CREs that alter the pattern of motor neuron differentiation in neural tissue. These findings establish quantitative principles for engineering synthetic regulatory elements with programmable signal responses to rewire genetic circuits and control stem cell differentiation, providing a basis for understanding developmental gene regulation and designing therapeutic gene expression systems.

developmental biology↗

bioRxiv2024

Illuminating morphogen and patterning dynamics with optogenetic control of morphogen production

Cells use dynamic spatial and temporal cues to instruct cell fate decisions during development. Morphogens are key examples, where the concentration and duration of morphogen exposure produce distinct cell fates that drive tissue patterning. Studying the dynamics of these processes has been challenging. Here, we establish an optogenetic system for morphogen production that enables the investigation of developmental patterning in vitro. Using a tunable light-inducible gene expression system, we generate long-range Shh gradients that pattern neural progenitors into spatially distinct progenitor domains mimicking the spatial arrangement of neural progenitors found in vivo during vertebrate neural tube development. With this system, we investigate how biochemical features of Shh and the presence of morphogen-interacting proteins affect the patterning length scale. We measure tissue clearance rates, revealing that Shh has an extracellular half-life of about 1h, and we probe how the level and duration of morphogen exposure govern the acquisition and maintenance of cell fates. The rate of Shh turnover is substantially faster than the downstream gene expression dynamics, indicating that the gradient is continually renewed during patterning. Together the optogenetic approach establishes a simple experimental system for the quantitative interrogation of morphogen patterning. Controlling morphogen dynamics in a reproducible manner provides a framework to dissect the interplay between biochemical cues, the biophysics of gradient formation, and the transcriptional programmes underlying developmental patterning.

developmental biology↗

bioRxiv2022

Optogenetic closed-loop feedback control of the unfolded protein response optimizes protein production

In biotechnological protein production processes, the onset of protein unfolding at high gene expression levels leads to diminishing production yields and reduced efficiency. Here we show that in silico closed-loop optogenetic feedback control of the unfolded protein response (UPR) clamps gene expression rates at intermediate near-optimal values, leading to significantly improved product titers. Specifically, in a fully-automated custom-built 1L-photobioreactor, we used a cybergenetic control system to steer the level of UPR in yeast to a desired set-point by optogenetically modulating the expression of a hard-to-fold protein based on real-time feedback measurements of the UPR, resulting in 60% higher product titers. This proof-of-concept study paves the way for advanced optimal biotechnology production strategies that diverge from and complement current strategies employing constitutive overexpression or genetically hardwired circuits.

bioengineering↗

bioRxiv2021

Synthetic gene networks recapitulate dynamic signal decoding and differential gene expression

Cells live in constantly changing environments and employ dynamic signaling pathways to transduce information about the signals they encounter. However, the mechanisms by which dynamic signals are decoded into appropriate gene expression patterns remain poorly understood. Here, we devise networked optogenetic pathways that achieve novel dynamic signal processing functions that recapitulate cellular information processing. Exploiting light-responsive transcriptional regulators with differing response kinetics, we build a falling-edge pulse-detector and show that this circuit can be employed to demultiplex dynamically encoded signals. We combine this demultiplexer with dCas9-based gene networks to construct pulsatile-signal filters and decoders. Applying information theory, we show that dynamic multiplexing significantly increases the information transmission capacity from signal to gene expression state. Finally, we use dynamic multiplexing for precise multidimensional regulation of a heterologous metabolic pathway. Our results elucidate design principles of dynamic information processing and provide original synthetic systems capable of decoding complex signals for biotechnological applications.

synthetic biology↗

bioRxiv2019

Cell-in-the-loop pattern formation with optogenetically emulated cell-to-cell signaling

1Designing and implementing synthetic biological pattern formation remains a challenge due to underlying theoretical complexity as well as the difficulty of engineering multicellular networks bio-chemically. Here, we introduce a \"cell-in-the-loop\" approach where living cells interact through in silico signaling, establishing a new testbed to interrogate theoretical principles when internal cell dynamics are incorporated rather than modeled. We present a theory that offers an easy-to-use test to predict the emergence of contrasting patterns in gene expression among laterally inhibiting cells. Guided by the theory, we experimentally demonstrated spontaneous checkerboard patterning in an optogenetic setup where cell-to-cell signaling was emulated with light inputs calculated in silico from real-time gene expression measurements. The scheme successfully produced spontaneous, persistent checkerboard patterns for systems of sixteen patches, in quantitative agreement with theoretical predictions. Our research highlights how tools from dynamical systems theory may inform our understanding of patterning, and illustrates the potential of cell-in-the-loop for engineering synthetic multicellular systems.

synthetic biology↗

bioRxiv2018

Tuning gene expression variability and multi-gene regulation by dynamic transcription factor control

Many natural transcription factors are regulated in a pulsatile fashion, but it remains unknown whether synthetic gene expression systems can benefit from such dynamic regulation. Using a fast-acting, light-responsive transcription factor in Saccharomyces cerevisiae, we show that dynamic pulsatile signals reduce cell-to-cell variability in gene expression. We then show that by encoding such signals into a single input, expression mean and variability can be precisely and independently tuned. Further, we construct a light-responsive promoter library and demonstrate how pulsatile signaling also enables graded multi-gene regulation at fixed expression ratios, despite differences in promoter dose-response characteristics. Pulsatile regulation can thus lead to highly beneficial functional behaviors in synthetic biological systems, which previously required laborious optimization of genetic parts or complex construction of synthetic gene networks.

synthetic biology↗

bioRxiv2017

Optogenetic Single-Cell Control Of Transcription Achieves Mrna Tunability And Reduced Variability

The study of gene expression at the single-cell level has exposed the importance of stochasticity for the behavior of cellular systems. Research on cellular variability has mostly relied on observing expression either in response to natural stimuli or to constant gene regulators. However, the ability to probe cells individually can lead to a deeper understanding of the underlying process. Here, we propose an experimental platform for optogenetic feedback control of individual cells. It consists of a digital micromirror device that, coupled to a microscope, can target light-responsive cells with individualized illumination profiles, thereby exploiting the good spatial resolution of optogenetic induction. Together with an automated software pipeline for segmentation, quantification and tracking of single cells, the platform enables independent and real-time control of numerous cells. We demonstrate our platform by regulating transcription in over a hundred yeast cells simultaneously, while achieving tunability of mRNA abundance. Using a novel technique to measure extrinsic variation, we further show that single cell feedback regulation of this highly stochastic process achieves a 10-fold reduction of extrinsic variation in nascent mRNA over population control, with superior control loop properties. Our platform establishes a new, flexible method for studying transcriptional dynamics in single cells.

synthetic biology↗