Project description:Purpose: dissolved oxygen (DO) level is an important factor that could significantly influence microorganisms’ growth, maintenance, metabolism and product yield. The goals of this study are to do comparative analysis on Corynebacterium glutamicum transcriptome in response to the change of dissolved oxygen in bioreactor, find the critical pathways and genes.
Project description:Purpose:dissolved oxygen (DO) level is an important factor that could significantly influence microorganisms’ growth, maintenance, metabolism and product yield.The goals of this study are to comparative analysis on Corynebacterium glutamicum transcriptome in response to expression of eGFP under the change of dissolved oxygen in bioreactor,find the critical pathways and genes.
Project description:Microarray transcriptional profiling was utilized to observe the global cellular state correlates of the physiological responses to temperature and oxygen changes. We provide evidence for the existence of correlations of Escherichia coli transcriptional responses to temperature and oxygen perturbations-precisely mirroring the co-occurrence of these parameters upon transitions between the outside world and the mammalian gastrointestinal-tract. Keywords: time course Physiological perturbations were carried out under a controlled environment in the context of bioreactor (Bioflo 110, New Brunswick Scientific) growth. Thermoelectric sensors and heaters were used to shift temperature profiles between 25 C and 37 C, and polarographic dissolved oxygen sensors (Mettler Toledo) and nitrogen gas was used to rapidly change oxygen saturation between anaerobic (0% dissolved oxygen) and aerobic (16-21% dissolved oxygen) condition. The cultures were maintained in exponential phase (O.D.600 0.2-0.4) through controlled dilution, where fresh media is pumped in and spent media is pumped out at a controlled rate. Prior to the perturbations, cells were maintained in the pre-transition environment for at least eight generations (8-24 hours). All experiments were performed in duplicate.
Project description:To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation. Four independent biological samples are included in this study. Two batches of cells were subjected to 20 minutes of hypoxia in a bioreactor; two cell batches were highly aerated.
Project description:To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation.
Project description:The exploration of scale-down models to imitate the influence of large scale bioreactor inhomogeneities on cellular metabolism is a topic with increasing relevance. While gradients of substrates, pH, or dissolved oxygen are often investigated, oscillating CO2/HCO3- levels, a typical scenario in large industrial bioreactors, is rarely addressed. Hereby, we investigate the metabolic and transcriptional response in Corynebacterium glutamicum wild type as well as the impact on L-lysine production in a model strain exposed to pCO2 gradients of (75-315) mbar. A novel three-compartment cascade bioreactor system was developed and characterized that offers high flexibility for installing gradients and residence times to mimic industrial-relevant conditions, and provides the potential of accurate carbon balancing. The phenomenological analysis of cascade fermentations imposed to the pCO2 gradients at industry-relevant residence times of about 3.55 min did not significantly impair the process performance, with growth and product formation being similar to control conditions. However, transcriptional analysis disclosed up to 66 differentially expressed genes already after 3.55min under stimulus exposure, with the overall change in gene expression directly correlateable to the pCO2 gradient intensity and the residence time of the cells.
Project description:Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2×106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6×106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.
Project description:Oxygen additions play a critical role in winemaking. However, few studies have focused on how this oxygen affects yeast metabolism and physiology in wine making conditions. We performed microarrays to unveil the oxygen response in wine making conditions. We extracted RNA from nitrogen-limited chemostats, simulating wine making conditions, sparged with nitrogen and 1%, 5% and 20% oxygen-nitrogen mixtures to achieve different dissolved oxygen levels representative of those found during wine making. These correspond to 0, 0.8, 4 and 11 micromolar dissolved oxygen
Project description:Antibiotic resistance genes (ARGs) and virulence genes (VGs) associated with bacterial pathogens are of great concern in WWTPs, while current knowledge of their profiles and co-occurrence patterns in different time intervals is barely sufficient. Moreover, the impacts of treatment process on ARG/VGs diversity also remain clear. To this end, this study was launched to address the differences of the ARG/VGs diversity between an oxidation ditch (OD) and an membrane bioreactor (MBR) and the co-occurrence patterns in different time intervals using a functional gene array-GeoChip.
Project description:Commonly used monolayer cell cultures lack the capacity to provide a physiologically relevant environment for cell culture in terms of cell-cell architecture, extracellular matrix composition, and spatiotemporal delivery of key growth factors and small molecules, such as oxygen. Here, we describe a three-dimensional (3D) approach to cell culture in vitro, utilizing a bioreactor system designed to control oxygenation of 3D cancer cell cultures, in order to better mimic tumor microenvironments observed in vivo. We found transcriptomic differences in breast and ovarian cancer cell cultures grown in traditional monolayer cultures as compared to cultures grown in a Matrigel three-dimensional matrix. We also investigated the transcriptomes of 3D cultures grown in 21% O2, 3% O2, and a gradient of 3% O2 to 0% O2 using our bioreactor system. By controlling oxygen delivery, we observed differences in cell growth morphology and transcriptome regulation under the three conditions.