Control of gene expression on the transcription level is vital in synthetic biology for the construction of complex and sophisticated tools. To achieve such control, synthetic genetic parts must be developed that can enable such control. Although much recent progress has been made in transcription factor-based biosensors for workhorse organisms, non-model organisms such as Clostridium still lack tools for rapid and customizable gene expression control. Clostridium species are biotechnologically relevant, but do not have a large number of transcription regulation devices, reducing the speed of their engineering. In this work, we show two systems for transcriptional control in bacterial systems. First, we demonstrate a Cas12a-based CRISPRi gene repression system for the tuning of biosynthetic pathways. Cas12a CRISPR effector proteins are better suited for use in Clostridium due to the genus’ AT-rich genomes and the corresponding simple and T-rich protospacer adjacent motif. We demonstrate in Clostridium pasteurianum tunable repression based on proximity to regulation elements, strand, and number of targeted sites through reporter genes, transcription levels, and redistribution of carbon flux. Second, we demonstrate optimization of transcription factor (TF) DNA binding sites through both a rationale and high-throughput naïve approach (sort-seq). We engineer a set of engineered product-responsive transcription factor regulated promoters (biosensors) for real-time single cell monitoring of butanol accumulation. We demonstrate tunability of gene expression though altering the number, location, and sequence of the transcription factor binding sites. Additionally, we show transcription factor-operator binding energy plays a key factor in the tunability of such systems. Biophysical characteristics between the TF-operator are evaluated using surface plasmon resonance (SPR). This work can enable rational strategies to edit the dynamic range of transcription factor-based biosensors.

Genetic devices for controlled gene expression in Clostridium

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.