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ALGORITHMIC DESIGN OF INTERCELLULAR GENETIC CIRCUITS TO PROGRAM DIVERSE MICROBIAL CONSORTIA

Abstract
Multicellular transcriptional regulatory networks control and coordinate dynamic responses in microbial consortia and are prevalent in natural systems to distribute metabolic and regulatory functions. However, this approach has yet to be widely adopted for engineering applications. To achieve coordinated multicellular responses, intercellular communication is required. Molecular intercellular signaling by homoserine lactones (HSLs) is prevalent in natural gram-negative bacterial quorum sensing and has been used in diverse applications. Nevertheless, a lack of well-characterized, modular components and natural signal crosstalk of HSL sensors have hindered the predictive design of multicellular regulatory networks for transcriptional programming. Here, we present an approach and comprehensive set of characterized DNA components to design multicellular genetic circuits in bacterial consortia. We develop standardized cell-cell logic gates for intercellular signaling that utilize five LuxR-type quorum sensors and signal attenuation gates comprised of characterized HSL-degrading enzymes for reversible and dynamic temporal signaling between bacterial cells. The transfer functions for all gates are characterized in standard relative promoter units to facilitate automated genetic circuit design using a scalable signal matching algorithm. The algorithm was validated for the design of multicellular genetic circuits in Escherichia coli consortia. Multicellular genetic circuits were designed to implement combinatorial logic and sequential logic for temporal transcriptional programming. Next, we applied targeted protein engineering to systematically and quantitatively elucidate the sequence-specificity relationship of a LuxR-type regulator and mitigate signal crosstalk. We designed and built a pooled combinatorial saturation mutagenesis library comprising 9,486 LasR protein variants by mutating the β5 sheet in its protein sequence. We performed high-throughput screening assays for the pooled library of sensor designs by fluorescence activated cell sorting and next-generation sequencing to quantify the activation of each to cognate and noncognate HSL signals. We identified 559 LasR sensors (5.7% of the library) with up to 60.6-fold improvement in signal specificity for the cognate HSL and negligible activation by the noncognate HSL. We uncovered previously unknown positions that confer signal specificity and prevalent mutational epistasis. Lastly, we sought to establish molecular intercellular signaling between diverse bacterial species. While gram-positive bacteria are not known to use HSL quorum sensing in nature, we engineered synthetic HSL sensors for gram-positive Bacillus subtilis using LuxR-type regulators and synthetic promoters. Using combinatorial promoter designs, screening, and design of experiments, we elucidated the effects of promoter elements and regulator expression for HSL-mediated activation in B. subtilis. This first set of HSL sensors for a gram-positive bacterium achieved at least 20.2-fold activation by each of the four HSL signals. This work demonstrates the algorithmic design of multicellular gene regulatory networks and an approach to effectively engineer highly orthogonal HSL sensors for parallel intercellular signaling, while also expanding their utility to diverse bacteria and thereby paving the way toward the development of programmable diverse bacterial consortia.
Type
campusfive
article
dissertation
Date
2023-09-01
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http://creativecommons.org/licenses/by-nc/4.0/
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2024-09-01T00:00:00-07:00
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