Project description:Thermal proteome profiling of E. coli lysate treated with fosfomycin (0.2 mg/ml). Untargeted proteomics with microflow method and with 60 min gradient.
Project description:Biotechnology has transformed the production of various chemicals and pharmaceuticals due to its efficient and selective processes, but it is inherently limited by its use of live cells as 'biocatalysts.' Cell-free expression (CFE) systems, which use a protein lysate isolated from whole cells, have the potential to overcome these challenges and broaden the scope of biomanufacturing. Implementation of CFE systems at scale will require determining clear markers of lysate activity and developing supplementation approaches that compensate for potential variability across batches and experimental protocols. Towards this goal, we use metabolomics to relate lysate preparation and performance to metabolic activity. We show that lysate processing affects the metabolite makeup of lysates, and that lysate metabolite levels change over the course of a CFE reaction regardless of whether a target compound is produced. Finally, we use this information to develop ways to standardize lysate activity and to design an improved CFE system.
Project description:The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state <i>via</i> lysate preincubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.
Project description:The gene expression profile of E. coli K-12 MG1655 grown in minimal medium treated with 0.12 mg/L of the biocide triclosan has been analysed using whole genome oligonucleotide microarrays. "Control" RNA was isolated from three independently grown 50ml MOPS minimal media cultures of E. coli K-12 MG1655. “Test” RNA was isolated from three independently grown 50ml MOPS minimal cultures of E. coli K-12 MG1655, to which was added 0.12 mg/L of triclosan after reaching mid-logarithmic growth phase (OD600 ~ 0.7 +/- 0.02). Keywords: dose response
Project description:We performed two dimensional thermal proteome profiling (2D-TPP) in Escherichia coli mutants to measure changes in abundance and thermal stability.
Project description:We utilize ribosome profiling to directly monitor translation in E. coli at 30 °C and investigate how this changes after 10-20 minutes of heat shock at 42 °C. Translation is controlled by the interplay of several RNA hybridization processes, which are expected to be temperature sensitive. We observe that translation efficiencies are robustly maintained after thermal heat shock and after mimicking the heat shock response transcriptional program at 30 °C.
Project description:Expression profile of E. coli BW25113 grown under standard laboratory atmosphere with a fine particulate matter (PM2.5) concentration of 17 mg m-3, under urban polluted atmosphere with a PM2.5 of 230 mg m-3 or under diesel exhaust atmosphere with a PM2.5 of 613 mg m-3. Expression profile of the diesel exhaust atmosphere-adapted E. coli strain T56-1 grown under diesel exhaust atmosphere.