(102b) High-Throughput Approaches for Engineering Tunable Gene Expression Regulation in Non-Model Bacteria
AIChE Annual Meeting
Monday, November 11, 2019 - 8:18am to 8:36am
In this work, we have developed two systems for transcriptional control in bacterial systems. First, we demonstrate optimization of transcription factor (TF) DNA binding sites through both a rationale and high-throughput naÃ¯ve 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 strategy is further demonstrated in which an activating TF was converted into a transcriptional repressor by relocating operating binding sites near or overlapping the RNAP binding site. This work can enable rational strategies to edit the dynamic range of transcription factor-based biosensors. Second, we have developed a regulatable CRISPRi gene repression for the fine-tuning of biosynthetic pathways using Cas12a effector proteins, which are better suited for use in Clostridium due to the genusâ 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. We propose a set of heuristics for such control as well as compare single versus multiplexed repression.