Novel genetic engineering tools for functional alteration of mammalian gut microbiomes

The gut microbiome is an integral component of the human body that plays a role in many physiological processes. Dysbiosis, an imbalance of the microbiome, has been associated with disease states including inflammatory bowel disease, type II diabetes, and obesity, and moreover, contributes to the pathogenesis of these states. Understanding the functional mechanisms governing microbial ecology and microbe-host interactions is essential to understanding the microbiome’s role in health and disease. However, at present, functional genetic studies of diverse natural mammalian gut microbiomes remain challenging, due to a lack of genetic tools for bacteria outside of a handful of well-studied model organisms. Altering the metagenome of a complex microbial community requires novel platform technologies for genetic engineering which can operate in a generalized fashion across many different host organisms. In this thesis, I present two novel genetic tools designed for genetic modification of bacterial communities. The first, the Cas-Transposon platform, is a host-independent targeted genome editing tool that utilizes programmable, targeted transposases to mediate site-specific gene insertions into user-defined loci. The Himar1 transposase naturally inserts transposases into random TA dinucleotides in a genome, but when fused to the dCas9 RNA-guided, DNA-binding protein, the fusion protein Himar1-dCas9 targets transposon insertions to a single TA site. The activity of Himar1-dCas9 was characterized using in vitro experiments, demonstrating that site-specific transposition is dependent on guide RNA (gRNA) orientation relative to the target site and the sequence surrounding the target site, but robust to variations in DNA and protein concentration, presence of background DNA, and temperature. We additionally showed that the Cas-Transposon platform is capable of performing site-specific transposition into a plasmid in vivo in E. coli, although further optimization of the system may be necessary to effect site-specific transposition into a genomic locus. The Himar1-dCas9 protein is the first example of a transposase that inserts transposons into locations programmable by an RNA, making it a novel tool for gene insertion and knockout in potentially any organism, without relying on DNA repair by a host cell. Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC) is an approach to directly modify gut bacteria in their native habitat by harnessing naturally occurring horizontal gene transfer activity to deliver engineered DNA. Because many gut bacteria are difficult to cultivate and thus difficult to genetically manipulate in the laboratory, MAGIC uses donor bacteria, delivered directly into the gut environment, to conjugate mobile vectors bearing engineered genetic payloads. Using payloads with selectable markers, we identified organisms across 4 major phyla of gut bacteria that were amenable to genetic modification with libraries of conjugative vectors we created. Using a lab-adapted E. coli strain as a donor, we achieved transient expression of the engineered payload in the microbiome. We also demonstrated that engineered native gut bacteria containing conjugative vectors could be deployed back into the gut to stably recolonize and mediate secondary transfer of the payload into other microbes, potentially enabling long-term infiltration of the payload into the metagenome. The results from this study suggest that both short-term and long-term genetic alteration of the metagenome are possible by choosing different donors, and that the MAGIC platform could enable development of more diverse microbial chasses for synthetic biology applications. MAGIC could also be used to create personalized engineered probiotics for diagnostic or therapeutic applications. In Chapter 4 of this thesis, we explored the targeted use of MAGIC to genetically modify Segmented Filamentous Bacteria, a gut commensal that is important for immune regulation but recalcitrant to in vitro cultivation. The Cas-Transposon and MAGIC technologies expand our capabilities in the areas of targeted genome editing and gene delivery into bacteria, respectively. Together, they form a suite of complementary approaches to genetically engineer undomesticated gut commensal bacteria and probe the functional genetic networks in the gut microbiome, which will enhance our understanding of microbiome ecology and host-microbiome interactions. In addition, the expanded range of genetic manipulations made possible by these tools may enable production of more diverse, perhaps personalized, probiotics containing engineered functions, such as sensing disease markers or drug delivery

Livestock and meat industry trends and restructuring technology for the meat industry in Taiwan, R.O.C.

Call number: LD2668 .R4 ASI 1988 C54Master of ScienceAnimal Sciences and Industr

Proprioceptive Coupling within Motor Neurons Drives C. Elegans Forward Locomotion

Locomotion requires coordinated motor activity throughout an animal’s body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement.Chemistry and Chemical BiologyPhysic

Novel genetic engineering tools for functional alteration of mammalian gut microbiomes

The gut microbiome is an integral component of the human body that plays a role in many physiological processes. Dysbiosis, an imbalance of the microbiome, has been associated with disease states including inflammatory bowel disease, type II diabetes, and obesity, and moreover, contributes to the pathogenesis of these states. Understanding the functional mechanisms governing microbial ecology and microbe-host interactions is essential to understanding the microbiome’s role in health and disease. However, at present, functional genetic studies of diverse natural mammalian gut microbiomes remain challenging, due to a lack of genetic tools for bacteria outside of a handful of well-studied model organisms. Altering the metagenome of a complex microbial community requires novel platform technologies for genetic engineering which can operate in a generalized fashion across many different host organisms. In this thesis, I present two novel genetic tools designed for genetic modification of bacterial communities. The first, the Cas-Transposon platform, is a host-independent targeted genome editing tool that utilizes programmable, targeted transposases to mediate site-specific gene insertions into user-defined loci. The Himar1 transposase naturally inserts transposases into random TA dinucleotides in a genome, but when fused to the dCas9 RNA-guided, DNA-binding protein, the fusion protein Himar1-dCas9 targets transposon insertions to a single TA site. The activity of Himar1-dCas9 was characterized using in vitro experiments, demonstrating that site-specific transposition is dependent on guide RNA (gRNA) orientation relative to the target site and the sequence surrounding the target site, but robust to variations in DNA and protein concentration, presence of background DNA, and temperature. We additionally showed that the Cas-Transposon platform is capable of performing site-specific transposition into a plasmid in vivo in E. coli, although further optimization of the system may be necessary to effect site-specific transposition into a genomic locus. The Himar1-dCas9 protein is the first example of a transposase that inserts transposons into locations programmable by an RNA, making it a novel tool for gene insertion and knockout in potentially any organism, without relying on DNA repair by a host cell. Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC) is an approach to directly modify gut bacteria in their native habitat by harnessing naturally occurring horizontal gene transfer activity to deliver engineered DNA. Because many gut bacteria are difficult to cultivate and thus difficult to genetically manipulate in the laboratory, MAGIC uses donor bacteria, delivered directly into the gut environment, to conjugate mobile vectors bearing engineered genetic payloads. Using payloads with selectable markers, we identified organisms across 4 major phyla of gut bacteria that were amenable to genetic modification with libraries of conjugative vectors we created. Using a lab-adapted E. coli strain as a donor, we achieved transient expression of the engineered payload in the microbiome. We also demonstrated that engineered native gut bacteria containing conjugative vectors could be deployed back into the gut to stably recolonize and mediate secondary transfer of the payload into other microbes, potentially enabling long-term infiltration of the payload into the metagenome. The results from this study suggest that both short-term and long-term genetic alteration of the metagenome are possible by choosing different donors, and that the MAGIC platform could enable development of more diverse microbial chasses for synthetic biology applications. MAGIC could also be used to create personalized engineered probiotics for diagnostic or therapeutic applications. In Chapter 4 of this thesis, we explored the targeted use of MAGIC to genetically modify Segmented Filamentous Bacteria, a gut commensal that is important for immune regulation but recalcitrant to in vitro cultivation. The Cas-Transposon and MAGIC technologies expand our capabilities in the areas of targeted genome editing and gene delivery into bacteria, respectively. Together, they form a suite of complementary approaches to genetically engineer undomesticated gut commensal bacteria and probe the functional genetic networks in the gut microbiome, which will enhance our understanding of microbiome ecology and host-microbiome interactions. In addition, the expanded range of genetic manipulations made possible by these tools may enable production of more diverse, perhaps personalized, probiotics containing engineered functions, such as sensing disease markers or drug delivery

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

Abstract Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology

Multiplex transcriptional characterizations across diverse bacterial species using cell‐free systems

Abstract Cell‐free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell‐free measurements is limited by the use of channel‐limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell‐Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual‐species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell‐free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology

Proprioceptive Coupling within Motor Neurons Drives C. elegans Forward Locomotion

Locomotion requires coordinated motor activity throughout an animal@s body. In both vertebrates and invertebrates, chains of coupled central pattern generators (CPGs) are commonly evoked to explain local rhythmic behaviors. In C. elegans, we report that proprioception within the motor circuit is responsible for propagating and coordinating rhythmic undulatory waves from head to tail during forward movement. Proprioceptive coupling between adjacent body regions transduces rhythmic movement initiated near the head into bending waves driven along the body by a chain of reflexes. Using optogenetics and calcium imaging to manipulate and monitor motor circuit activity of moving C. elegans held in microfluidic devices, we found that the B-type cholinergic motor neurons transduce the proprioceptive signal. In C. elegans, a sensorimotor feedback loop operating within a specific type of motor neuron both drives and organizes body movement