Biology subjects

Lucks, J.

Publications and source records attributed to Lucks, J..

5 recordsLinked to original sources

bioRxiv2018

Distinct timescales of RNA regulators enable the construction of a genetic pulse generator

To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.

synthetic biology↗

bioRxiv2017

Engineering a Functional small RNA Negative Autoregulation Network with Model-guided Design

RNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal noise. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs. Using parameter sensitivity analysis, we design a simple set of experiments that allow us to accurately predict NAR function in TX-TL. We transfer successful network designs in vivo and show that our sRNA transcriptional network reduces both network response time and noise in steady-state gene expression. This work broadens our ability to construct increasingly sophisticated RNA genetic networks with predictable function.

synthetic biology↗

bioRxiv2017

Computational design of Small Transcription Activating RNAs (STARs) for versatile and dynamic gene regulation

A longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this goal is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design - allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to [~]9000-fold gene activation. We then demonstrate the versatility of RNA-based transcription control by showing the broad utility of STARs - from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.

synthetic biology↗

bioRxiv2017

Characterizing The Structure-Function Relationship Of A Naturally-Occurring RNA Thermometer

A wide number of bacteria have been found to govern virulence and heat shock responses using temperature-sensing RNAs known as RNA thermometers. A prime example is the agsA thermometer known to regulate the production of the AgsA heat shock protein in Salmonella enterica using a \"fourU\" structural motif. Using the SHAPE-Seq RNA structure-probing method in vivo and in vitro, we found that the regulator functions by a subtle shift in equilibrium RNA structure populations that lead to a partial melting of the helix containing the ribosome binding site. We also demonstrate that ribosome binding to the agsA mRNA causes changes to the thermometer structure that appear to facilitate thermometer helix unwinding. These results demonstrate how subtle RNA structural changes can govern gene expression and illuminate the function of an important bacterial regulatory motif.

biochemistry↗

bioRxiv2017

Distributed Biotin-Streptavidin Transcription Roadblocks for Mapping Cotranscriptional RNA Folding

RNA molecules fold cotranscriptionally as they emerge from RNA polymerase. Cotranscriptional folding is an important process for proper RNA structure formation as the order of folding can determine an RNA molecules structure, and thus its functional properties. Despite its fundamental importance, the experimental study of RNA cotranscriptional folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structures at nucleotide resolution during transcription. We previously developed cotranscriptional selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-seq) to simultaneously probe all of the intermediate structures an RNA molecule transitions through during transcription elongation. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent streptavidin roadblocking strategy to simplify the preparation of roadblocking transcription templates. We determine the fundamental properties of streptavidin roadblocks and show that randomly distributed streptavidin roadblocks can be used in cotranscriptional SHAPE-Seq experiments to measure the Bacillus cereus crcB fluoride riboswitch folding pathway. Comparison of EcoRIE111Q and streptavidin roadblocks in cotranscriptional SHAPE-Seq data shows that both strategies identify the same RNA structural transitions related to the riboswitch decision-making process. Finally, we propose guidelines to leverage the complementary strengths of each transcription roadblock for use in studying cotranscriptional folding.

molecular biology↗