Biology subjects

Gorochowski, T. E.

Publications and source records attributed to Gorochowski, T. E..

3 recordsLinked to original sources

bioRxiv2018

Absolute quantification of translational regulation and burden using combined sequencing approaches

Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a parts natural context and allow only a few parameters to be monitored concurrently. Here, we develop an approach that combines ribosome profiling (Ribo-seq) with quantitative RNA sequencing (RNA-seq) to enable the high-throughput characterization of genetic parts controlling translation in absolute units. We simultaneously measure 743 translation initiation rates and 746 termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a {sigma}32-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches towards a comprehensive and quantitative understanding of gene regulation and burden in living cells.

synthetic biology↗

bioRxiv2017

Organisation of feed-forward loop motifs reveals architectural principles in natural and engineered networks

Network motifs are significantly expressed sub-graphs that have been proposed as building blocks for natural and engineered networks. Detailed functional analysis has been performed for many types of motif in isolation, but less is known about how motifs work together to perform complex tasks. To address this issue we measure the aggregation of network motifs via methods that extract precisely how these structures are connected. Applying this approach to a broad spectrum of networked systems and focusing on the widespread feed-forward loop motif, we uncover striking differences in motif organisation. The types of connection are often highly constrained, differ between domains, and clearly capture architectural principles. We show how this information can be used to effectively predict functionally important nodes in the metabolic network of Escherichia coli. Our findings have implications for understanding how networked systems are constructed from motif parts and elucidates constraints that guide their evolution.

bioinformatics↗

bioRxiv2017

Burden-driven feedback control of gene expression

Cells use feedback regulation to ensure robust growth despite fluctuating demands on resources and different environmental conditions. Yet the expression of foreign proteins from engineered constructs is an unnatural burden on resources that cells are not adapted for. Here we combined multiplex RNAseq with an in vivo assay to reveal the major transcriptional changes in two E. coli strains when a set of inducible synthetic constructs are expressed. We identified that native promoters related to the heat-shock response activate expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a CRISPR/dCas9-based feedback regulation system that automatically adjusts synthetic construct expression in response to burden. Cells equipped with this general-use controller maintain capacity for native gene expression to ensure robust growth and as such outperform unregulated cells at protein yields in batch production. This engineered feedback is the first example of a universal, burden-based biomolecular control system and is modular, tuneable and portable.

synthetic biology↗