Progress 09/01/23 to 08/31/24
Outputs
Target Audience:Target audiences include scientists, engineers, and eventually biotechnology entrepreneurs who are interested in synthetic biology methodologies for biocontainment of genetically engineered (GE) microbes. For example, scientists and engineers have been developing GE probiotics to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers. Another example is that engineers are interested in developing microbes to do bioremediation in the environment. However, administered GE microbes are expected to be released into the environment. The consequences of such releases are difficult to assess, preventing federal regulatory agencies from approving those potential applications. This biosafety concern can be addressed by implementing our synthetic biology methodologies for biocontainment of GE microbes, if successfully developed.? Changes/Problems:Dr. Moon left Washington University in St. Louis and joined J. Craig Venter Institute. What opportunities for training and professional development has the project provided?Multiple graduate students (e.g., Yuefeng Ma and Alamri Nadiyah) have been trained (total FTE = 1 with multiple months for each student) by participating in various activities related to the project. How have the results been disseminated to communities of interest?The results have been disseminated by 9 papers and many presentations during the reporting period (Sep. 2023 to Aug. 2024). Invited talks given by Dr. Moon - 47 times Contributed talks and poster presentations by Dr. Moon and the lab members - 17 times What do you plan to do during the next reporting period to accomplish the goals?Developing aromatic 'Passcode' kill switches to sustainably remediate phenolic compounds from environments We will continue to develop aromatic 'Passcode' kill switches to sustainably remediate phenolic compounds from environments. Specifically, we will transformRhodococcus opacusPD630 (R. opacus), whichhas the natural ability to tolerate and consume toxic phenolic compounds, into a bioremediation platform for phenolic pollutant removal with the ability to trigger a kill switch that achieves self-killing after accomplishing the remediation task. We will continue to develop anAromatic Passcode Kill Switch (APKS)by using a previously characterized aromatic responsive promoter and the transcriptional repressor TetR and other transcription factors. This circuit allowsR. opacusto perform phenolic compound degradation and self-destruct in a self-controlled and programmable manner by automatically sensing the changes in phenolic compound concentrations without any additional inducers or manual interventions. After optimization, our engineered strain can completely remove the phenolic compounds from environments with killing ability when the APKS circuit is initiated.
Impacts
What was accomplished under these goals?
Our team has published 9 papers, which acknowledge the USDA support, and multiple papers are under review in journals at this moment. [1] C Xi, Y Ma, M Amrofell andTS Moon. Manipulating the molecular specificity of transcriptional biosensors for tryptophan metabolites and analogs.Cell Reports Physical Science, 5, 102211, 2024 Tryptophan and its metabolites, produced by the gut microbiota, are pivotal for human/animal physiological and mental health. Yet, quantifying these structurally similar compounds with high specificity remains a challenge, hindering point-of-care diagnostics and targeted therapeutic interventions. Leveraging the innate specificity and adaptability of biological systems, we present a biosensing approach capable of identifying specific metabolites in complex contexts with minimal cross-activity. This study introduces a generalizable strategy that combines evolutionary analysis, key ligand-binding residue identification, and mutagenesis scanning to pinpoint ligand-specific transcription factor variants. Furthermore, we uncover regulatory mechanisms within uncharacterized ligand-binding domains, whether in homodimer interfaces or monomers, through structural prediction and ligand docking. Notably, our ''plug-and-play'' strategy broadens the detection spectrum, enabling the exclusive bio-sensing of indole-3-acetic acid (an auxin, also a plant hormone), tryptamine, indole-3-pyruvic acid, and other tryptophan derivatives in engineered probiotics. This groundwork paves the way to create highly specific transcriptional biosensors for potential clinical, agricultural, and industrial use. [2]TS Moon. Probiotic and microbiota engineering for practical applications.Current Opinion in Food Science, 56, 101130, 2024 In this perspective article, Dr. Moon discusses the potential of engineering probiotics and microbial consortia for detecting and curing gut diseases, solving food shortages, enabling sustainable bioproduction, mitigating pollution, and addressing the climate crisis. [3] C Xi, J Diao andTS Moon. Advances in ligand-specific biosensing for structurally similar molecules.Cell Systems, 14 (12), 1024-1043, 2023 In this invited review, we discuss the recent advances in the development of specific biosensors for potential practical applications, including agricultural and environmental applications. [4] Y Ma, A Manna andTS Moon. Advances in engineering genetic circuits for microbial biocontainment.Current Opinion in Systems Biology, 36, 100483, 2024 The development of biocontainment strategies is ongoing, including the use of kill switches, auxotrophy, and stringent response circuits, to control the viability of genetically engineered microbes (GEMs). In this review we discuss the applications and future directions of genetic circuits for microbial biocontainment strategies. [5] B Barsola, S Saklani, D Pathania, P Kumari, S Sonu, S Rustagi, P Singh, P Raizada,TS Moon, and A Kaushik. Exploring bio-nanomaterials as antibiotic allies to combat antimicrobial resistance.Biofabrication, 16, 042007, 2024 In this review, we discuss bio-nanomaterials as ways to effectively address antibiotic or antimicrobial drug resistance issues. [6] ER Aurand,TS Moon, NR Buan, KV Solomon, M Köpke & EBRC Technical Roadmapping Working Group. Addressing the climate crisis through engineering biology.npj Climate Action, 3, 9, 2024 In this perspective, we discuss climate crisis and approaches to address it using engineering biology approaches. [7]TS Moon. Earth: Extinguishing anthropogenic risks through harmonization.New Biotechnology, 80, 69-71, 2024 In this perspective article, four recommendations to address climate issues are discussed, emphasizing paradigm changes as well as the use of safe and sustainable technologies. [8] MB Amrofell, S Rengarajan, S Vo, ESR Tovar, L LoBello, G Dantas andTS Moon. EngineeringE. colistrains using antibiotic resistance gene-free plasmids.Cell Reports Methods, 3, 100669, 2023 Plasmids have long been used in biological studies as a simple way of genetically engineering microbes, but researchers typically rely on antibiotic resistance for continued maintenance of plasmids. Current methods to circumvent this reliance are laborious or do not fully remove the antibiotic resistance cassette. In our work, we established the utility of multiple differentE. coliDH10B auxotrophic cloning strains for constructing plasmids that substitute dual auxotrophic gene and essential gene markers for an antibiotic resistance cassette. Using these plasmids, we also showed we can easily transform different engineered strains ofE. coliNissle 1917 (EcN), a common chassis for microbial therapeutics and diagnostics, as well asE. coliMG1655, the laboratory ''wild-type''E. colistrain, which are missing the genomic copies of both the essential gene and auxotrophic gene. Notably, we showed that the engineered Nissle strains are capable of plasmid maintenance for a month of repeated culturing as well as in the mouse gut, demonstrating the strong potential for a variety of applications in non-controlled, real-world environments without causing antibiotic resistance spread via horizontal gene transfer. In short, our work describes a total pipeline for antibiotic resistance gene-free plasmid (ARGFP)-based cloning and maintenance, potentially changing the laboratory practice and paradigm. [9]TS Moon. Seven governing principles in biology.Frontiers in Synthetic Biology, 1:1296513, 2023 In this perspective article, Dr. Moon discusses seven governing principles in biology.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
C Xi, Y Ma, M Amrofell and TS Moon. Manipulating the molecular specificity of transcriptional biosensors for tryptophan metabolites and analogs. Cell Reports Physical Science, 5, 102211, 2024
Tryptophan and its metabolites, produced by the gut microbiota, are pivotal for human/animal physiological and mental health. Yet, quantifying these structurally similar compounds with high specificity remains a challenge, hindering point-of-care diagnostics and targeted therapeutic interventions. Leveraging the innate specificity and adaptability of biological systems, we present a biosensing approach capable of identifying specific metabolites in complex contexts with minimal cross-activity. This study introduces a generalizable strategy that combines evolutionary analysis, key ligand-binding residue identification, and mutagenesis scanning to pinpoint ligand-specific transcription factor variants. Furthermore, we uncover regulatory mechanisms within uncharacterized ligand-binding domains, whether in homodimer interfaces or monomers, through structural prediction and ligand docking. Notably, our plug-and-play strategy broadens the detection spectrum, enabling the exclusive bio-sensing of indole-3-acetic acid (an auxin, also a plant hormone), tryptamine, indole-3-pyruvic acid, and other tryptophan derivatives in engineered probiotics. This groundwork paves the way to create highly specific transcriptional biosensors for potential clinical, agricultural, and industrial use.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
TS Moon. Probiotic and microbiota engineering for practical applications. Current Opinion in Food Science, 56, 101130, 2024
In this perspective article, Dr. Moon discusses the potential of engineering probiotics and microbial consortia for detecting and curing gut diseases, solving food shortages, enabling sustainable bioproduction, mitigating pollution, and addressing the climate crisis.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
C Xi, J Diao and TS Moon. Advances in ligand-specific biosensing for structurally similar molecules. Cell Systems, 14 (12), 1024-1043, 2023
In this invited review, we discuss the recent advances in the development of specific biosensors for potential practical applications, including agricultural and environmental applications.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Y Ma, A Manna and TS Moon. Advances in engineering genetic circuits for microbial biocontainment. Current Opinion in Systems Biology, 36, 100483, 2024
The development of biocontainment strategies is ongoing, including the use of kill switches, auxotrophy, and stringent response circuits, to control the viability of genetically engineered microbes (GEMs). In this review we discuss the applications and future directions of genetic circuits for microbial biocontainment strategies.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
B Barsola, S Saklani, D Pathania, P Kumari, S Sonu, S Rustagi, P Singh, P Raizada, TS Moon, and A Kaushik. Exploring bio-nanomaterials as antibiotic allies to combat antimicrobial resistance. Biofabrication, 16, 042007, 2024
In this review, we discuss bio-nanomaterials as ways to effectively address antibiotic or antimicrobial drug resistance issues.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
ER Aurand, TS Moon, NR Buan, KV Solomon, M K�pke & EBRC Technical Roadmapping Working Group. Addressing the climate crisis through engineering biology. npj Climate Action, 3, 9, 2024
In this perspective, we discuss climate crisis and approaches to address it using engineering biology approaches.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
TS Moon. Earth: Extinguishing anthropogenic risks through harmonization. New Biotechnology, 80, 69-71, 2024
In this perspective article, four recommendations to address climate issues are discussed, emphasizing paradigm changes as well as the use of safe and sustainable technologies.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
MB Amrofell, S Rengarajan, S Vo, ESR Tovar, L LoBello, G Dantas and TS Moon. Engineering E. coli strains using antibiotic resistance gene-free plasmids. Cell Reports Methods, 3, 100669, 2023
Plasmids have long been used in biological studies as a simple way of genetically engineering microbes, but researchers typically rely on antibiotic resistance for continued maintenance of plasmids. Current methods to circumvent this reliance are laborious or do not fully remove the antibiotic resistance cassette. In our work, we established the utility of multiple different E. coli DH10B auxotrophic cloning strains for constructing plasmids that substitute dual auxotrophic gene and essential gene markers for an antibiotic resistance cassette. Using these plasmids, we also showed we can easily transform different engineered strains of E. coli Nissle 1917 (EcN), a common chassis for microbial therapeutics and diagnostics, as well as E. coli MG1655, the laboratory wild-type E. coli strain, which are missing the genomic copies of both the essential gene and auxotrophic gene. Notably, we showed that the engineered Nissle strains are capable of plasmid maintenance for a month of repeated culturing as well as in the mouse gut, demonstrating the strong potential for a variety of applications in non-controlled, real-world environments without causing antibiotic resistance spread via horizontal gene transfer. In short, our work describes a total pipeline for antibiotic resistance gene-free plasmid (ARGFP)-based cloning and maintenance, potentially changing the laboratory practice and paradigm.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
TS Moon. Seven governing principles in biology. Frontiers in Synthetic Biology, 1:1296513, 2023
In this perspective article, Dr. Moon discusses seven governing principles in biology.
|
Progress 09/01/22 to 08/31/23
Outputs
Target Audience:Target audiences include scientists, engineers, and eventually biotechnology entrepreneurs who are interested in synthetic biology methodologies for the biocontainment of genetically engineered (GE) microbes. For example, scientists and engineers have been developing GE probiotics to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers. Another example is that engineers are interested in developing microbes to do bioremediation in the environment. However, administered GE microbes are expected to be released into the environment. The consequences of such releases are difficult to assess, preventing federal regulatory agencies from approving those potential applications. This biosafety concern can be addressed by implementing our synthetic biology methodologies for the biocontainment of GE microbes, if successfully developed. Changes/Problems:The lab could not hire new project members for two years, delaying project expenditures. Despite this issue, the lab has made tremendous progress thanks to the current members. What opportunities for training and professional development has the project provided?Multiple graduate students (Yuefeng Ma and Alamri Nadiyah) and postdoctoral researchers (Jinjin Diao and Abhijit Manna) have been trained (total FTE = 1 with a few months for each trainee) by participating in various activities related to the project, including presentations in international conferences. How have the results been disseminated to communities of interest?The results have been disseminated by seven papers (including ones under review or in preparation) and many conference presentations for the reporting year (Sep. 2022 to Aug. 2023). For example, many presentations have been given by PI Moon: 68 times in 2022 and 48 times in 2023 What do you plan to do during the next reporting period to accomplish the goals?[1] We will continue to develop "suicide" circuits in genetically engineered (GE)Escherichia coliNissle (EcN) that will be engineered to produce antimicrobial peptides. The focus will be engineering the module to produce antimicrobial peptides. This probiotic EcN strain can be potentially used as a "pathogen killer" in the animal gut, which commits "suicide" when released into the environment. [2] We will continue to develop "suicide" circuits in a consortium of GER. opacusstrains that will be engineered to consume toxic aromatic compounds. The focus will be engineering the module to sense and consume toxic aromatic compounds. In addition, we will develop genetic tools to engineer the consortia ofR. opacusstrains or soil bacteria. This consortium of GER. opacusstrains can be potentially used as pollutant degraders in a contaminated site, which commit "suicide" when the cleanup is completed.
Impacts
What was accomplished under these goals?
Our team has published one paper, and six papers are under review or in preparation. [1] AG Rottinghaus, S. Vo and TS Moon. Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia.PNAS, 120 (1) e2213154120, 2022 Microbes naturally coexist in complex, multi-strain communities. However, extracting individual microbes from and specifically manipulating the composition of these consortia remain challenging. The sequence-specific nature of CRISPR guide RNAs can be leveraged to accurately differentiate microorganisms and facilitate the creation of tools that can achieve these tasks. In this project, we developed a computational program, ssCRISPR, which designs strain-specific CRISPR guide RNA sequences with user-specified target strains, protected strains, and guide RNA properties. We experimentally verify the accuracy of the strain specificity predictions in bothEscherichia coliandPseudomonasspp. and show that up to three nucleotide mismatches are often required to ensure perfect specificity. As a proof of concept, we applied the program to two novel applications: the isolation of specific microbes from consortia through plasmid transformations and the removal of specific microbes from consortia through liposome packaged CRISPR antimicrobials. For strain purification, we utilize gRNAs designed to target and kill all microbes in a consortium except the specific microbe to be isolated. For strain elimination, we utilize gRNAs designed to target only the unwanted microbe while protecting all other strains in the community. To our knowledge, this is the first demonstration of both applications. Our study demonstrates that the liposome packaged CRISPR payload can be delivered to microbes in diverse ecosystems, including intestines, blood, lungs, and soil, which has vast implications in designing strain-specific antimicrobials and combating the growing concern of antibiotic- and bacteriocide-resistant microbes. [2] JJ Diao, YF Ma, A. Manna and TS Moon.Developing aromatic 'Passcode' kill switches to sustainably remediate phenolic compounds from waters. In preparation. Discharge of the phenolic compounds without pretreatment would lead to serious adverse impacts on the ecosystem because of their inherent toxic, mutagenic, teratogenic, and carcinogenic nature. With the development of biotechnologies, more and more microbes with the metabolic capacity to degrade phenolic compounds have been isolated and sequenced, which opens a new biotechnological path for remediating phenolic compounds. However,to avoid the technical hurdles of the potential practical application of genetically engineered microorganisms for phenolic compound bioremediation, the development of a highly efficient and genetically stable kill switch circuit is necessary. In this project, we attempted to transformRhodococcus opacusPD630 (hereafter,R. opacus)- a Gram-positive soil bacterium that has the natural ability to tolerate and consume toxic phenolic compounds - into a bioremediation platform for phenolic pollutant removal with the ability to trigger a microbial biocontainment system that achieves self-killing after accomplishing the remediation task. Rather than manipulating and assembling heterologous pathways, the potent endogenous aromatic catabolic network was taken as a native phenolic compound degradation module. Then, we developed anAromatic Passcode Kill Switch (APKS)by using a previously characterized aromatic responsive promoter and the transcriptional repressor TetR. This circuit allowsR. opacusto perform phenolic compound degradation and self-destruct in a self-controlled and programmable manner by automatically sensing the changes in phenolic compound concentrations without any additional inducers or manual interventions.After optimization, our engineered strain could completely remove the phenolic compounds from waters with 100% killing efficiency when the APKS circuit was initiated, irrespective of the environmental conditions (i.e., standard minimal medium or model ecosystem). Moreover, the APKS circuit exhibited significant long-term stability, with 100% killing efficiency maintained after 60 days (~500 generations) of continuous growthin vitroeven in the nutritional deficient condition. Overall, these results demonstrate that with the development of robust kill switch circuits, microbes can be transformed into contained bioremediation platforms, which have the feasibility to be applied in open environments. [3] RL Mancinelli, TS Moon et al. Biosafety commonalities between synthetic biology and planetary contamination protection. In preparation In this perspective article, PI Moon and others discuss biosafety issues, including biocontainment of genetically engineered organisms. [4] MB Amrofell, S. Vo and TS Moon. EngineeringE. colistrains using antibiotic resistance gene-free plasmids. Cell Reports Methods. Under review Plasmids have long been used in biological studies as a simple way of genetically engineering microbes, yet researchers typically rely on antibiotic resistance for continued maintenance of plasmids. Current methods to circumvent this reliance are laborious or do not fully remove the antibiotic resistance cassette. In our work, we established the utility of two differentE. coliDH10B auxotrophic cloning strains for constructing plasmids that substitute dual auxotrophic gene and essential gene markers for an antibiotic resistance cassette. Using these plasmids, we showed we can easily transform two different engineered strains ofE. coliNissle 1917 (EcN), a common chassis for microbial therapeutics and diagnostics, which are missing the genomic copies of both the essential gene and auxotrophic gene. Ultimately, we demonstrate that these Nissle strains will maintain these plasmids after repeatedly culturing them for a month in vitro and will work in a real-world environment such as an animal gut. In short, our work describes a total pipeline for antibiotic resistance gene-free plasmid (ARGFP) cloning and maintenance. This pipeline has important ramifications in the field of microbiology. Not only will it enable plasmid maintenance in non-laboratory environments, such as the gastrointestinal tract or soil, but it can also prevent the spread of antibiotic resistance in those environments. In addition, as recently suggested by PI Moon (TS Moon, Trends in Biotechnology, 40, p1405-1414), a huge amount of DNA containing antibiotic resistance cassettes has been released by research laboratories into the environment, requiring new laboratory practices for disposing of biological waste all over the world. While we focus on two EcN-based examples of a working strain, EcN is a medically important strain, and our pipeline serves as a blueprint for developing ARGFP maintenance in other microbial species. Our work does not currently include a demonstration in a fully non-controlled environment, other than the animal tests, due to the regulatory policy, but we think the technology will be broadly applicable in the future. [5] ER Aurand, TS Moon, NR Buan, KV Solomon, M Köpke and EBRC Technical Roadmapping Working Group. Addressing the Climate Crisis Through Engineering Biology. Under review In this perspective article, we discuss synthetic biology solutions to climate crisis and pollution. [6] Y Ma, A Manna and TS Moon. Advances in engineering genetic circuits for microbial biocontainment. Current Opinion in Systems Biology. Under review. In this invited review, we discuss recent advances in biocontainment strategies. [7] C Xi, J Diao and TS Moon. Advances in ligand-specific biosensing for structurally similar molecules. Cell Systems. Under review. In this invited review, we discuss recent advances in the development of specific biosensors for potential practical applications, including agricultural and environmental applications.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
AG Rottinghaus, S. Vo and TS Moon. Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia. PNAS, 120 (1) e2213154120, 2022
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
JJ Diao, YF Ma, A. Manna and TS Moon. Developing aromatic Passcode kill switches to sustainably remediate phenolic compounds from waters. In preparation.
- Type:
Journal Articles
Status:
Other
Year Published:
2023
Citation:
RL Mancinelli, TS Moon et al. Biosafety commonalities between synthetic biology and planetary contamination protection. In preparation
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
MB Amrofell, S. Vo and TS Moon. Engineering E. coli strains using antibiotic resistance gene-free plasmids. Cell Reports Methods. Under review
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
ER Aurand, TS Moon, NR Buan, KV Solomon, M K�pke and EBRC Technical Roadmapping Working Group. Addressing the Climate Crisis Through Engineering Biology. Under review
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Y Ma, A Manna and TS Moon. Advances in engineering genetic circuits for microbial biocontainment. Current Opinion in Systems Biology. Under review.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
C Xi, J Diao and TS Moon. Advances in ligand-specific biosensing for structurally similar molecules. Cell Systems. Under review.
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Target audiences include scientists, engineers, and eventually biotechnology entrepreneurs who are interested in synthetic biology methodologies for biocontainment of genetically engineered (GE) microbes. For example, scientists and engineers have been developing GE probiotics to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers. However, administered GE probiotics are expected to be released together with feces into the environment. The consequences of such releases are difficult to assess, preventing federal regulatory agencies from approving those potential applications. This biosafety concern can be addressed by implementing our synthetic biology methodologies for biocontainment of GE microbes, if successfully developed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Multiple graduate students have been trained (total FTE = 1 with a few months for each student) byparticipating in various activities related to the project. PI Moon serves as the SynBYSS Founding Chair - Synthetic Biology Young Speaker Series A weekly, virtual, multi-year seminar series in which a global thought leader or a synthetic biology pioneer gives an opening talk (~5 min), followed by a ~45 min talk and Q&A given by a rising untenured faculty or a faculty candidate; since 8/26/2021 (at least until 2023) Global audiences watch the recorded, weekly YouTube video (with 89,351 total views as of 11/14/2022). https://www.youtube.com/channel/UCTkyl4Uw1NlpuLCeP4Zi8aA How have the results been disseminated to communities of interest?The results have been disseminated by 5 papers and many presentations for one year (Sep. 2021 to Aug. 2022). Invited talks given by PI Moon - 46times Contributed talks and poster presentations by PI Moon and others - 24 times What do you plan to do during the next reporting period to accomplish the goals?[1] We will demonstrate "suicide" circuits in GE Escherichia coli Nissle (EcN) that will be engineered to produce antimicrobial peptides. The focus will be engineering the module to produce antimicrobial peptides. This probiotic EcN strain can be potentially used as a "pathogen killer" in the animal gut, which commits "suicide" when released into the environment. [2] We will develop "suicide" circuits in a consortium of GE R. opacus strains that will be engineered to consume toxic aromatic compounds. The focus will be engineering the module to sense and consume toxic aromatic compounds. In addition, we will develop genetic tools to engineer the consortia of R. opacus strains or soil bacteria. This consortium of GE R. opacus strains can be potentially used as pollutant degraders in a contaminated site, which commit "suicide" when the cleanup is completed.
Impacts
What was accomplished under these goals?
The team members have published five articles, and two articles are under review in top journals such as PNAS and Nature Communications. [1]AG Rottinghaus, A Ferreiro, SRS Fishbein, G Dantas and TS Moon. Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications. 13, 672 (2022) Kill switch systems have been developed in bacteria, but kill switches are highly susceptible to mutational inactivation due to the selective pressure, leading to escape mutant populations and incomplete killing (e.g., Chan et al., Nat Chem Biol 2016). To address this, we leveraged parallel strategies to maximize kill switch efficacy and maintain genetic stability, including the use of multiple induction modalities (both chemical and environmental), functional redundancies within the kill switch circuit, an antibiotic-independent plasmid maintenance system, genomic knockouts of SOS response genes to mitigate mutagenesis, and provision of intra-niche competition by a closely related non-kill switch strain to better mimic the real environment. With these combined approaches, we demonstrate complete elimination of kill switch strains within the guts of mice upon chemical induction, and complete biocontainment after excretion with temperature induction. We further demonstrate genetic stability of our system both in vitro (at least 224 generations or 28 days of cultures) and in vivo (at least 8 days under selection). Applying this modular, generalizable kill switch platform to soil bacteria, we are expanding our biocontainment strategy to develop genetically stable kill switches in soil bacteria such as Rhodococcus and Pseudomonas strains. In addition, to gain insights into the behavior of kill-switch strains under real environments, we are testing the recently developed kill-switch strain using media containing real environment samples. [2] TS Moon and A Rottinghaus, U.S. Provisional App No. 63/328,628 (07-APR-2022) [3] AG Rottinghaus, S. Vo and TS Moon. Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia. PNAS. Under review Modifying microbial consortia with strain-specificity is critical for maintaining stable and healthy microbiota. We have developed and validated a novel computational program, ssCRISPR, which designs strain-specific CRISPR guide RNAs (gRNAs) that can be utilized to modify complex consortia. As a proof of concept, we applied the program to two novel applications: the isolation of specific microbes from consortia through plasmid transformations and the removal of specific microbes from consortia through liposome packaged CRISPR antimicrobials. To our knowledge, this is the first demonstration of both applications. One provisional patent has been filed, and one paper is currently under review. ssCRISPR gRNAs can be used in diverse applications, including improving the health of livestock, plants, and humans, identifying and isolating microbes with unique characteristics, investigating the roles of microbial communities, and tailoring microbiota for improved functions. Using ssCRISPR, we showed a simple plasmid transformation workflow to isolate individual microbes from a consortium. This technique shortens and simplifies microbial isolation techniques, which currently involve complex tailored media and serial culture systems, and it allows for the discovery of microbes with novel characteristics. Next, we demonstrated a novel strain-specific antimicrobial by packaging ssCRISPR-designed CRISPR cassettes in liposomes. These liposomes can fuse with and deliver the CRISPR payload to microbes in diverse ecosystems, including intestines, blood, lungs, and soil. This new technique has vast implications in designing strain-specific antimicrobials and combating the growing concern of antibiotic- and bacteriocide-resistant microbes. [4] TS Moon. Engineering the future through synthetic biology. Biotechnol. Bioproc. Engin. Invited Perspective. In press (2022) In this perspective article, PI Moon discusses his vision to enable the transition of Synthetic Biology from fundamental study to translational practice. [5] TS Moon. SynMADE: Synthetic Microbiota Across Diverse Ecosystems. Trends in Biotechnology. Invited Opinion Article.(2022) PI Moon discusses future directions to develop microbiota as a biomanufacturing host. Specifically, he proposes that we can develop the soil microbial community itself as a huge bioreactor. Ultimately, researchers will provide a generalizable system that enables us to understand microbial consortium's interaction and metabolism at diverse temporal and spatial scales to address global problems, including the climate crisis, food inequality, waste issue, and sustainable bioproduction. [6]MB Amrofell, S. Vo and TS Moon. Engineering E. coli strains using antibiotic resistance gene-free plasmids. Nature Communications. Under review Plasmids have long been used in biological study as a simple way of genetically engineering microbes, yet researchers typically rely on antibiotic resistance for continued maintenance of plasmids. Current methods to circumvent this reliance are laborious or do not fully remove the antibiotic resistance cassette. In our work, we established the utility of two different E. coli DH10B auxotrophic cloning strains for constructing plasmids that substitute dual auxotrophic gene and essential gene markers for an antibiotic resistance cassette. Using these plasmids, we showed we can easily transform two different engineered strains of E. coli Nissle 1917 (EcN), a common chassis for microbial therapeutics and diagnostics, which are missing the genomic copies of both the essential gene and auxotrophic gene. Ultimately, we demonstrate that these Nissle strains will maintain these plasmids after repeatedly culturing them for a month. In short, our work describes a total pipeline for antibiotic resistance gene-free plasmid (ARGFP) cloning and maintenance. This pipeline has important ramifications in the field of microbiology. Not only will it enable plasmid maintenance in non-laboratory environments, such as the gastrointestinal tract or soil, but it can also prevent the spread of antibiotic resistance in those environments. In addition, as recently suggested (TS Moon, Trends in Biotechnology, 40, p1405-1414), a huge amount of DNA containing antibiotic resistance cassettes has been released by research laboratories into the environment, requiring new laboratory practices for disposing of biological waste all over the world. [7]J Diao, R Carr and TS Moon. Deciphering the metabolic and regulatory networks of aromatic catabolism using synthetic biology tools. Nature Commun. Biol. 5, 1109 (2022) This work provides new, fundamental information about the complex interplay of transcriptional elements in regulating the catabolism of lignin-derived aromatics in the gram-positive soil bacterium, Rhodococcus opacus. We also demonstrate that the hierarchical utilization of different aromatics can be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways. This work is a significant achievement, given the lack of comprehensive, molecule-level, mechanistic study despite a plethora of omics-based systems biology reports. [8]AG Rottinghaus, C Xi, MB Amrofell, H Yi and TS Moon. Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals. Cell Systems. 13, 204-214.e4 (2022) This work lays the ground and framework for developing sensors. The computational approach is generalizable to develop others sensors necessary for this USDA project.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
AG Rottinghaus, A Ferreiro, SRS Fishbein, G Dantas and TS Moon. Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications. 13, 672 (2022)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
J Diao, R Carr and TS Moon. Deciphering the metabolic and regulatory networks of aromatic catabolism using synthetic biology tools. Nature Commun. Biol. 5, 1109 (2022)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
AG Rottinghaus, C Xi, MB Amrofell, H Yi and TS Moon+. Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals. Cell Systems. 13, 204-214.e4 (2022)
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
TS Moon. SynMADE: Synthetic Microbiota Across Diverse Ecosystems. Trends in Biotechnology. 40, 1405-1414 (2022)
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
TS Moon. Engineering the future through synthetic biology. Biotechnol. Bioproc. Engin.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
AG Rottinghaus, S. Vo and TS Moon. Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia. PNAS.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
MB Amrofell, S. Vo and TS Moon+. Engineering E. coli strains using antibiotic resistance gene-free plasmids. Nature Commun.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Target audiences include scientists, engineers, and eventually biotechnology entrepreneurs who are interested in synthetic biology methodologies for biocontainment of genetically engineered (GE) microbes. For example, scientists and engineers have been developing GE probiotics to produce antimicrobial peptides in the gastrointestinal tract of livestock, providing new pathogen reduction strategies for farmers. However, administered GE probiotics are expected to be released together with feces into the environment. The consequences of such releases are difficult to assess, preventing federal regulatory agencies from approving those potential applications. This biosafety concern can be addressed by implementing our synthetic biology methodologies for biocontainment of GE microbes, if successfully developed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One PhD student has been trained by participating in various activities related to the project. The PI has advised one high-school iGEM team, broadening its impact. How have the results been disseminated to communities of interest?The PI has given more than 40 presentations in 2021 (more than 50 if presentations given by students and postdocs are included). In addition to these presentations, the team has published multiple papers to disseminate the results. What do you plan to do during the next reporting period to accomplish the goals?[1]We will demonstrate "suicide" circuits in GEEscherichia coliNissle (EcN) that will be engineered to produce antimicrobial peptides. The focus will be engineering the module toproduce antimicrobial peptides.This probiotic EcN strain can be potentially used as a "pathogen killer" in the animal gut, which commits "suicide" when released into the environment. [2]We will develop "suicide" circuits in a consortium of GER. opacusstrains that will be engineered to consume toxic aromatic compounds. Thefocus will be engineering the module to sense andconsume toxic aromatic compounds. In addition, we will develop genetic tools to engineer the consortia of R. opacusstrains or soil bacteria.This consortium of GER. opacusstrains can be potentially used as pollutant degraders in a contaminated site, which commit "suicide" when the cleanup is completed.
Impacts
What was accomplished under these goals?
As described in the publishedpapers or ones under review (see below), wedemonstrated "suicide" circuits in GEEscherichia coliNissle (EcN), developed application-relevant sensors, and engineeredR. opacusstrains. +, corresponding author J Diao, R Carr and TS Moon+. Deciphering the metabolic and regulatory networks of aromatic catabolism using synthetic biology tools. Under review AG Rottinghaus, A Ferreiro, SRS Fishbein, G Dantas+ and TS Moon+. Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications. Under review (minor revision) AG Rottinghaus, C Xi, MB Amrofell, H Yi and TS Moon+. Engineering probiotics for specific sensing of aromatic amino acids or neurochemicals. Cell Systems. Accepted (2021) DM DeLorenzo, J Diao, R Carr, Y Hu and TS Moon+. An improved CRISPR interference tool to engineer Rhodococcus opacus. ACS Synth. Biol. 10, 786-798 (2021)
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Rottinghaus et al., Engineering ligand-specific biosensors for aromatic amino acids and neurochemicals, Cell Systems
(2021), https://doi.org/10.1016/j.cels.2021.10.006
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
AG Rottinghaus, A Ferreiro, SRS Fishbein, G Dantas and TS Moon. Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications. Under review (minor revision)
- Type:
Journal Articles
Status:
Under Review
Year Published:
2022
Citation:
J Diao, R Carr and TS Moon. Deciphering the metabolic and regulatory networks of aromatic catabolism using synthetic biology tools. Under review
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
DM DeLorenzo, J Diao, R Carr, Y Hu and TS Moon. An improved CRISPR interference tool to engineer Rhodococcus opacus. ACS Synth. Biol. 10, 786798 (2021)
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