Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography

Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures

Methods for Detecting Microbial Methane Production and Consumption by Gas Chromatography

Methane is an energy-dense fuel but is also a greenhouse gas 25 times more detrimental to the environment than CO2. Methane can be produced abiotically by serpentinization, chemically by Sabatier or Fisher-Tropsh chemistry, or biotically by microbes (Berndt et al., 1996; Horita and Berndt, 1999; Dry, 2002; Wolfe, 1982; Thauer, 1998; Metcalf et al., 2002). Methanogens are anaerobic archaea that grow by producing methane gas as a metabolic byproduct (Wolfe, 1982; Thauer, 1998). Our lab has developed and optimized three different gas chromatograph-utilizing assays to characterize methanogen metabolism (Catlett et al., 2015). Here we describe the end point and kinetic assays that can be used to measure methane production by methanogens or methane consumption by methanotrophic microbes. The protocols can be used for measuring methane production or consumption by microbial pure cultures or by enrichment cultures

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

An Investigation into a Methane Producing World: The Collective Work in Route to Terpene Production Platforms of Methanosarcina

Terpenes represent the largest class of natural compounds, which can be used in a wide range of applications from medicines to flavor additives. Current technologies are limited by non-environmentally friendly synthesis techniques (petroleum-based) or inadequate production values (native plant production). To solve this limitation, synthetic biologists have engineered microbes to become terpene production platforms. Although great success has come from the use of some of these platforms, these engineered organisms natively lack high flux through necessary terpene producing pathways. To rectify this problem, extensive metabolic engineering has to be completed in order to obtain industrial-relevant production levels. This dissertation looks to expand the microbial terpene platform idea through the genetic modification of an anaerobic, methane-producing archaeon whose metabolic substrates and byproducts may allow for improved terpene production than previously used microbial models. Methanosarcina is a genus of methanogens that natively produces high levels of prenyl precursors for cellular membrane synthesis, leading to the hypothesis that this pathway can be tapped for terpene production. To show the genus ability to produce high levels of a desired terpene, a modified genetic system was employed to genetically engineer Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro to express a functional isoprene synthase. Genetic modification of these strains by the addition of a single enzyme involved in isoprene biosynthesis results in high levels of isoprene production with negligible impact on microbial fitness. The isoprene produced is at levels comparable with existing isoprene producing microbes; but an advantage of Methanosarcina use is in feedstock as it can be readily grown on cheap or often free, waste products. To further the idea that Methanosarcina can be developed into a terpene production platform, strains overexpressing isoprenyl diphosphates synthase were created. Such strains showed increased lipid production compared to wild-type, indicating that high carbon flux is flowing through the terpene precursor pathway. This leads to the promising conclusion that Methanosarcina can be used as the desired platform system

An Investigation into a Methane Producing World: The Collective Work in Route to Terpene Production Platforms of Methanosarcina

Terpenes represent the largest class of natural compounds, which can be used in a wide range of applications from medicines to flavor additives. Current technologies are limited by non-environmentally friendly synthesis techniques (petroleum-based) or inadequate production values (native plant production). To solve this limitation, synthetic biologists have engineered microbes to become terpene production platforms. Although great success has come from the use of some of these platforms, these engineered organisms natively lack high flux through necessary terpene producing pathways. To rectify this problem, extensive metabolic engineering has to be completed in order to obtain industrial-relevant production levels. This dissertation looks to expand the microbial terpene platform idea through the genetic modification of an anaerobic, methane-producing archaeon whose metabolic substrates and byproducts may allow for improved terpene production than previously used microbial models. Methanosarcina is a genus of methanogens that natively produces high levels of prenyl precursors for cellular membrane synthesis, leading to the hypothesis that this pathway can be tapped for terpene production. To show the genus ability to produce high levels of a desired terpene, a modified genetic system was employed to genetically engineer Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro to express a functional isoprene synthase. Genetic modification of these strains by the addition of a single enzyme involved in isoprene biosynthesis results in high levels of isoprene production with negligible impact on microbial fitness. The isoprene produced is at levels comparable with existing isoprene producing microbes; but an advantage of Methanosarcina use is in feedstock as it can be readily grown on cheap or often free, waste products. To further the idea that Methanosarcina can be developed into a terpene production platform, strains overexpressing isoprenyl diphosphates synthase were created. Such strains showed increased lipid production compared to wild-type, indicating that high carbon flux is flowing through the terpene precursor pathway. This leads to the promising conclusion that Methanosarcina can be used as the desired platform system

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, Methanosarcina acetivorans

PRODUCTION OF ISOPRENE BY METHANE - PRODUCING ARCHAEA

Plasmid vectors and use of plasmid vectors in methods for producing methane and isoprene using Archaea are disclosed . Particularly, plasmid vectors that express isoprene synthase ( ispS ) are prepared and inserted into methanogens, such as Methanosarcina acetivorans, to allow for co - production of methane and isoprene. In one embodiment , the methods of the present disclosure can be used for wastewater management

Anaerobic Production of Isoprene by Engineered \u3ci\u3eMethanosarcina\u3c/i\u3e Species Archaea

Isoprene is a valuable petrochemical used for a wide variety of consumer goods, such as adhesives and synthetic rubber. We were able to achieve a high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina species methanogens, it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show that engineered methanogens direct up to 4% of total carbon flux to isoprene, demonstrating that methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria, and from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate that isoprene synthesis depletes membrane precursors and redirects electron flux, enabling isoprene to be a major metabolic product. Our results demonstrate that methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs relative to those for petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The “isoprenogen” strains we developed have potential to be used for industrial production of inexpensive renewable isoprene

PRODUCTION OF ISOPRENE BY METHANE - PRODUCING ARCHAEA

Plasmid vectors and use of plasmid vectors in methods for producing methane and isoprene using Archaea are disclosed . Particularly, plasmid vectors that express isoprene synthase ( ispS ) are prepared and inserted into methanogens, such as Methanosarcina acetivorans, to allow for co - production of methane and isoprene. In one embodiment , the methods of the present disclosure can be used for wastewater management

pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, Methanosarcina acetivorans