48 research outputs found
An integrated cell-free metabolic platform for protein production and synthetic biology
Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements
Metabolic investigation of host/pathogen interaction using MS2-infected Escherichia coli
<p>Abstract</p> <p>Background</p> <p>RNA viruses are responsible for a variety of illnesses among people, including but not limited to the common cold, the flu, HIV, and ebola. Developing new drugs and new strategies for treating diseases caused by these viruses can be an expensive and time-consuming process. Mathematical modeling may be used to elucidate host-pathogen interactions and highlight potential targets for drug development, as well providing the basis for optimizing patient treatment strategies. The purpose of this work was to determine whether a genome-scale modeling approach could be used to understand how metabolism is impacted by the host-pathogen interaction during a viral infection. <it>Escherichia coli</it>/MS2 was used as the host-pathogen model system as MS2 is easy to work with, harmless to humans, but shares many features with eukaryotic viruses. In addition, the genome-scale metabolic model of <it>E. coli </it>is the most comprehensive model at this time.</p> <p>Results</p> <p>Employing a metabolic modeling strategy known as "flux balance analysis" coupled with experimental studies, we were able to predict how viral infection would alter bacterial metabolism. Based on our simulations, we predicted that cell growth and biosynthesis of the cell wall would be halted. Furthermore, we predicted a substantial increase in metabolic activity of the pentose phosphate pathway as a means to enhance viral biosynthesis, while a break down in the citric acid cycle was predicted. Also, no changes were predicted in the glycolytic pathway.</p> <p>Conclusions</p> <p>Through our approach, we have developed a technique of modeling virus-infected host metabolism and have investigated the metabolic effects of viral infection. These studies may provide insight into how to design better drugs. They also illustrate the potential of extending such metabolic analysis to higher order organisms, including humans.</p
Microbial Maintenance: A Critical Review on Its Quantification
Microbial maintenance is an important concept in microbiology. Its quantification, however, is a subject of continuous debate, which seems to be caused by (1) its definition, which includes nongrowth components other than maintenance; (2) the existence of partly overlapping concepts; (3) the evolution of variables as constants; and (4) the neglect of cell death in microbial dynamics. The two historically most important parameters describing maintenance, the specific maintenance rate and the maintenance coefficient, are based on partly different nongrowth components. There is thus no constant relation between these parameters and previous equations on this subject are wrong. In addition, the partial overlap between these parameters does not allow the use of a simple combination of these parameters. This also applies for combinations of a threshold concentration with one of the other estimates of maintenance. Maintenance estimates should ideally explicitly describe each nongrowth component. A conceptual model is introduced that describes their relative importance and reconciles the various concepts and definitions. The sensitivity of maintenance on underlying components was analyzed and indicated that overall maintenance depends nonlinearly on relative death rates, relative growth rates, growth yield, and endogenous metabolism. This quantitative sensitivity analysis explains the felt need to develop growth-dependent adaptations of existing maintenance parameters, and indicates the importance of distinguishing the various nongrowth components. Future experiments should verify the sensitivity of maintenance components under cellular and environmental conditions
The rumen microbial metagenome associated with high methane production in cattle
Acknowledgements The Rowett Institute of Nutrition and Health and SRUC are funded by the Rural and Environment Science and Analytical Services Division (RESAS) of the Scottish Government. The project was supported by Defra and the DA funded Agricultural Greenhouse Gas Inventory Research Platform, the Technology Strategy Board (Project No: TP 5903ā40240) and the Biotechnology and Biological Sciences Research Council (BBSRC; BB/J004243/1, BB/J004235/1). Our thanks are due to the excellent support staff at the SRUC Beef and Sheep Research Centre, Edinburgh, and to Silvia Ramos Garcia for help in interrogating the data. MW and RR contributed equally to the paper and should be considered as joint last authors.Peer reviewedPublisher PD