Project description:We were awarded a BBSRC grant about a year ago to undertake some affymetrix gene chip profiling of light and CO2 systemic signalling in Arabidopsis. The design of the proposed experiment is given below and the appropriate funding has been provided by the BBSRC. The aim of the project is to identify the temporal profile of those genes that respond to light and CO2 systemic signals in developing leaves. Moreover, as thes two signals have opposing effects on leaf development to ascertain whether they involve similar or parallel signalling pathways. The experiment is to examine the effect of exposing mature leaves to high CO2 or low light or both on the gene expression profile of developing leaves. We already have data for maize that changes in gene expression profile occur within 4h and that there are a variety of temporal responses that differ between individual gene transcripts. We have also demonstrated that Arabidopsis leaf development is altered by these systemmic signals and that lesions in the jasmonate and ethylene signalling pathways block these responses. Our experimental design is shown below:; We have 4 treatments and 7 timepoints. (0, 2, 4, 12, 24, 48, 96 h); We would sample from 5 individual plants that would be pooled for each RNA preparation. This would require 28 chips and this would include extra replication of the 0 time-point control (deemed by many as nessary). Experimental details:; All plants were germinated for 7 days under the following conditions:; Humax multi-purpose compost, ambient carbon dioxide (370 ppm) and ambient light (250 Âµmol/m/s), constant temperature of 20Â°C and a 10 h photoperiod (8 am until 6 pm). After a week the the seedlings were potted up into 104-cell plug trays for a further 2 weeks and then potted up into 10 cm pots and the bottom part of the signalling cuvette system attached (see Lake et al., Nature 10th May 2001 Vol. 411, pp 154). Twenty four, 4 week old plants, then had the top part of the signalling system attached, trapping leaf insertions 5-13. Humidified, ambient air was passed through them at 500 mls/min via an oil-free air compressor. The three target leaves (19-21) were then marked with non-toxic, acrylic paint. After a 24 h period (the plants were sealed into the cuvettes from 10 am until 10am) of adjustment, the experiment was started by harvesting the target leaves from 4 plants and immediately freezing the tissue in liquid nitrogen to give the 0 h sample before RNA extraction. The remaining 20 plants were divided into 4 groups of five and given one of the following treatments:; Ambient carbon dioxide/ambient light (Control) (A); Elevated carbon dioxide (750 ppm)/ambient light (E); Ambient carbon dioxide/low light (50 Âµmol/m/s) (AS); Elevated carbon dioxide/low light (ES); - For the Elevated CO2, elevated CO2 was pumped in using a CT room next door set to same temperature but with a CO2 cylinder inside and the same pump as used in the ambient room to supply the elevated CO2 laden, humidified air into the signalling room using rubber tubing. - Shade treatment consisted of neutral density filter (Cat. 210 0.6ND, Lee Filters) that had a hole cut in the middle to allow the middle developing leaves to grow through. A timecourse of 2, 4, 12, 24, 48 and 96 h were carried out each using a batch of 24 plants. This whole process was repeated with another batch of 24 plants at the same developmental stage to give a 2, 4, 12, 24, 48 and 96 hour sample from each of the four treatments. The whole timecourse was then repeated 4 times. For the mature leaves:; We had 8 chips left over so we devised this little experiment to assess the gene changes that were occurring in the enclosed, treated, mature leaves that were signalling the environment to the young developing leaves. Experimenter name = Simon Coupe; Experimenter phone = 0114 222 4115; Experimenter fax = 0114 222 0002; Experimenter institute = University of Sheffield; Experimenter address = Animal and Plant Sciences; Experimenter address = University of Sheffield; Experimenter address = Western Bank; Experimenter address = Sheffield; Experimenter zip/postal_code = S10 2TN; Experimenter country = UK Experiment Overall Design: 8 samples were used in this experiment

Project description:Escherichia coliÂ spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness. Expression differences of commonly shared genes (all four strains) andÂ E. coliÂ core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species. Four E.coli strains, laboratory strain K12 (MG1655), a commensal model strain (IAI1), a soil-isolated strain (TW11588-Clade IV), and a freshwater-isolated strain (TW09308âClade V) were used. Each strain was grown on a minimal growth medium (Ihssen and Egli, 2004) in three treatment modes: chemostat, batch, and starvation. Cells from batch culture were collected when reaching steady-state. For starvation, the medium flow was stopped during steady-state and bacteria were collected after 4âh.

Project description:In order to provide information about the gene expression response that occurs when cells experience a change in carbon source, succinate limited chemostat cultures of Methylobacterium extorquens AM1 were grown to and maintained at an OD of ~0.63, transferred to flasks and methanol was added. Cells were harvested for RNA extraction at time: 0 min, 10 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr post transition. At 30 min, a no methanol addition sample was extracted as a carbon starvation control. These data were used in conjunction with flux, enzymatic and metabolite measurements to assess the changes in central metabolism during this transition. Abstract from manuscript: When organisms experience environmental change, how does their metabolic network reset and adapt to the new condition? This study focused on the mechanisms of metabolic adaptation occurring during the transition from succinate to methanol growth by the methylotrophic bacterium Methylobacterium extorquens, analyzing changes in carbon flux, gene expression, metabolites and enzymatic activities over time. Initially, cells experienced metabolic imbalance with excretion of metabolites, changes in nucleotide levels and cessation of cell growth. Though assimilatory pathways were induced rapidly, a transient block in carbon flow to biomass synthesis occurred, and enzymatic assays suggested methylenetetrahydrofolate dehydrogenase as one control point. This “downstream priming” mechanism ensures that significant carbon flux through these pathways does not occur until they are fully induced, precluding the buildup of toxic intermediates. Most metabolites that are required for growth on both carbon sources did not change significantly, even though transcripts and enzymatic activities required for their production changed radically, underscoring the concept of metabolic setpoints. Gene expression in succinate limited chemostat cultures was compared to gene expression in cells transferred to flasks before and after methanol addition. As a control, a time = 0 sample (RNA prepared from cells harvested directly from the chemostat) was compared to a time = 0 sample immediately obtained after the cells were transferred to flasks, before methanol was added in order to identify changes due to flask transfer. A carbon starvation control was also done comparing expression from time = 0 (chemostat cells) to cells transferred to flasks for 30 min with no carbon source added. Two biological replicates each with two techinal replicates (dye swap) were analyzed for time = 0 (chemostat) vs 10 min, 30 min, 1 hr and 2 hr after methanol addition. One biological replicate with two technical replicates (dye swap) were analyzed for time = 0 (chemostat) vs time = 0 (flask transfer), and time = 0 (chemostat) vs time = 4 hr, 6 hr and 30 min no methanol addition.

Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness. The goal of the present study is to investigate the physiology of aerobic fully respiring S. cerevsiae at near-zero growth rates. Fundamental but industrially-relevant questions were addressed thanks to the design, implementation and study of aerobic retentostat cultivations enabling a rapid but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates.

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RNA-Seq of Pichia pastoris GS115 to determine the effect of inducing heterologous expression of human lysozyme (HuLy) variants

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