Browsing by Author "Chappell, James"
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Item A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria(Springer Nature, 2023) Liu, Baiyang; Samaniego, Christian Cuba; Bennett, Matthew R.; Franco, Elisa; Chappell, JamesA lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. Toward addressing this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore an RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.Item A split ribozyme that links detection of a native RNA to orthogonal protein outputs(Springer Nature, 2023) Gambill, Lauren; Staubus, August; Mo, Kim Wai; Ameruoso, Andrea; Chappell, JamesIndividual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.Item Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces(Oxford University Press, 2022) Ameruoso, Andrea; Villegas Kcam, Maria Claudia; Cohen, Katherine Piper; Chappell, JamesThe rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing the isolation and screening of novel chemicals. To address this, we describe a novel approach to activate silent BGCs through rewiring endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, we refine CRISPR interference (CRISPRi) and create CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. We then harness these tools to activate a silent BGC by perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery.Item Developing CRISPR tools for activating silent biosynthetic gene clusters in Streptomyces spp.(2022-09-06) Ameruoso, Andrea; Chappell, James; Bartel, BonnieThe rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing the isolation and screening of novel chemicals. To address this, I describe a novel approach to activate silent BGCs through rewiring of the endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, I refine CRISPR interference (CRISPRi) and create CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. I then harness these tools to activate a silent BGC by perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery.Item Embargo Development of ribozyme-derived RNA tools for engineering microbes and microbial consortia(2022-10-07) Gambill, Lauren Celeste; Chappell, JamesThe advent of next generation sequencing has illuminated the members present in microbial communities and we have begun to better characterize these diverse and complex communities by capturing both culturable and unculturable members. With this wealth of information in hand, we can now begin to think about adding control to these environments for numerous applications. Towards this goal, we need to know which members of a given community can be engineered and we need to be able to selectively control subpopulations of microbes within these communities using genetic programs. Here, we have developed two ribozyme-derived tools, which we call RENDR (Ribozyme-ENabled Detector of RNA) and RAM (Ribozyme-Addressable Memory). We built RENDR from the ground up, first showing that the cis-acting ribozyme functionally splices two exons together to enable translation of the transcript. We then split the ribozyme into two fragments and added complimentary RNA guides, which allowed us to probe a library of ribozyme split variants for functionality. The RNA guides were altered to bind to a third RNA, wherein design rules demonstrated the importance of RNA guide length on ribozyme splicing efficiency. Finally, we swapped the GFP output with colorimetric, gas-producing, and transcriptional regulator outputs, and demonstrated RENDR functionality across a diverse set of microbial hosts. With RAM, we first demonstrated functionality of the system and elucidated design rules by using a GFP splicing assay to analyze splicing efficiencies. We then targeted the RNA guide of RAM to 16S rRNA, conjugated a microbial consortium, and used DNA sequencing to determine which microbes were conjugated. These tools will help us to better understand how different conditions – like temperature and the presence of disinfectants – alter conjugation at the consortia level and how we might go about engineering microbes and microbial consortia to be more robust to negative perturbations in the community.Item Embargo Development of trans-acting RNA guided tools to control gene expression in bacteria(2022-11-08) Villegas Kcam, Maria Claudia; Chappell, JamesThe ability to control gene expression is a transformative capability that allows scientists to uncover gene function or induce valuable phenotypes and cell fates important for applications in biotechnology and biomedicine. We are specifically interested in the development of trans-acting regulators that can target non-synthetic sequences allowing for applications in endogenous pathways and high throughput screenings. CRISPR-Cas editors and regulators have provided an excellent toolbox for this purpose. However, current bacterial CRISPR activators (CRISPRa) present several challenges such as the lack of a global activation domain (AD) that can be used across bacteria and the restricted availability of functional binding sites in non-synthetic targets. To address these limitations we generated a highly modular CRISPRa platform where each element of the system (dCas9, sgRNA, AD, and target gene) can be easily modified, and thus we could screen for different CRISPRa designs. Initially, we identified a new AD for CRISPRa that we harnessed to explore different AD recruitment strategies and create the modular CRISPRa platform. Next, we evaluated the use of circularly permuted variants of the dCas9 protein to identify variants capable of activating gene expression from targeting positions previously identified as non-functional sites for CRISPRa. Additionally, we used the modular platform to create the first Type I-E CRISPRa system for bacteria and uncovered how its distinct properties differ from the previously used Type II- CRISPRa. With this study, we open the possibility for new CRISPRa systems harnessing different CRISPR systems to obtain distinct properties. Finally, we applied the CRISPRa system in Streptomyces venezuelae, a bacteria of interest for the production of new antibiotics. We characterized the CRISPRa system in S. venezuelae and targeted an endogenous pathway to induce the production of an antibiotic product. We demonstrate that the modular CRISPRa platform can be adapted to provide a more flexible and versatile tool that will expand our ability to activate and control bacterial genomes.Item Frontiers in Programming Living Systems: Hybrid Cell-Material Systems for Real-Time Sensing and Actuating(Rice University, 5/4/2024) Chappell, James; Frow, Emma; Kamat, Neha; Matthews, Kirstin; Montclare, Jin; Silberg, Jonathan; Sonkusale, Sameer; Tran, Lesa; Wilson, CoreyItem Rational engineering of a modular bacterial CRISPR-Cas activation platform with expanded target range(Rice University, 2020) Chappell, James; Department of BiosciencesCRISPR-Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR-Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.Item Rational engineering of a modular bacterial CRISPR–Cas activation platform with expanded target range(Oxford University Press, 2021) Villegas Kcam, Maria Claudia; Tsong, Annette J; Chappell, JamesCRISPR–Cas activator (CRISPRa) systems that selectively turn on transcription of a target gene are a potentially transformative technology for programming cellular function. While in eukaryotes versatile CRISPRa systems exist, in bacteria these systems suffer from a limited ability to activate different genes due to strict distance-dependent requirements of functional target binding sites, and require greater customization to optimize performance in different genetic and cellular contexts. To address this, we apply a rational protein engineering approach to create a new CRISPRa platform that is highly modular to allow for easy customization and has increased targeting flexibility through harnessing engineered Cas proteins. We first demonstrate that transcription activation domains can be recruited by CRISPR–Cas through noncovalent protein-protein interactions, which allows each component to be encoded on separate and easily interchangeable plasmid elements. We then exploit this modularity to rapidly screen a library of different activation domains, creating new systems with distinct regulatory properties. Furthermore, we demonstrate that by harnessing a library of circularly permuted Cas proteins, we can create CRISPRa systems that have different target binding site requirements, which together, allow for expanded target range.Item Embargo RNA-based portable genetic circuits and synthetic plasmids(2023-11-09) Liu, Baiyang; Chappell, JamesThe lack of modularity impedes the creation of synthetic biological systems at the same level of complexity as modern electronic systems, which can contain millions of logic gates. Recently, the development of novel RNA regulators poses an opportunity to overcome this challenge. In this thesis work, we aim to use RNA-based strategies to improve the modularity of biological systems on three different levels: chassis modularity, circuit modularity, and part modularity. Firstly, we verify the portability of Small Transcription Activating RNA (STAR) regulatory system in diverse Gram-negative bacteria. Then we break the chassis barrier by creating a regulatory RNA array design that allows for the construction of portable RNA-based circuits in multiple bacteria. Secondly, we propose an RNA compensation strategy to mitigate the retroactivity generated by the interconnection of multiple modules in RNA-based circuits. The insulation strategy is computationally proven to be effective not only in static modules but also in dynamic RNA-based circuits. Lastly, we develop synthetic plasmid origin of replication (SynOri) through the refactoring and reengineering of natural plasmid pMB1. The replication mechanism of pMB1 is replaced by pT181 RNA attenuator which allows the creation of modular plasmid library and plasmid replication-based circuits.