Expression profile of E. coli in response to exogenous addition of isopentenol
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ABSTRACT: Exogenous isopentenol was added to a culture of E. coli and the RNA expression response was measured using Nimblegen arrays. Genes highly upregulated were then subsequently overexpressed to improve the tolerance to isopentenol toxicity. In addition a subset of genes that improved the tolerance were also overexpressed in a producing strain, leading to improved production over the empty vector control. This work is described in Foo et al, Improving microbial bio-gasoline production in E. coli using tolerance engineering (in preparation) Six independent biological replicates were grown in M9 medium. 0.2% v/v isopentenol was added to 3 cultures while 3 cultures received no treatment, and 2.5 hr later total RNA was extracted. One sample from each culture was hybridized.
Project description:Whole genome gene expression level changes in E. coli DH1 exposed to 150 mM [C2mim]Cl in mid exponential growth, and compared to a non-exposed strain. Total RNA was isolated from three biological replicates for each condition: 150 mM [C2mim]Cl exposed at OD(600nm) 0.6, after 30 and 60 min, no exposure to [C2mim]Cl at OD(600 nm) 0.6 after 30 and 60 min.
Project description:Exogenous isopentenol was added to a culture of E. coli and the RNA expression response was measured using Nimblegen arrays. Genes highly upregulated were then subsequently overexpressed to improve the tolerance to isopentenol toxicity. In addition a subset of genes that improved the tolerance were also overexpressed in a producing strain, leading to improved production over the empty vector control. This work is described in Foo et al, Improving microbial bio-gasoline production in E. coli using tolerance engineering (in preparation)
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in LB and fed 10 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 2.5, 5, 7, and 29.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:n-Butanol has been proposed as an alternative biofuel to ethanol, and both Escherichia coli and Saccharomyces cerevisiae have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell wide studies were conducted at the transcript, protein and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicate that n-butanol stress has components common to other stress responses and includes perturbation in respiratory functions (nuo, cyo operons), oxidative stress (sodC, katG, yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, degP, cpxPR), metabolite transport (malE, opp operon) and biosynthesis. Inducible expression of the yqhD gene was found to improve the hostâs tolerance to exogenous n-butanol and confirms the role of this gene in coping with butanol stress. To survey for other potential candidates that may serve to improve host tolerance, mutant strains in several candidates which show changes at the transcript and protein levels were examined for sensitivity during butanol exposure. Chassis engineering based on these cues may be required in a high production titer, butanol-producing host. This comparison is between E. coli DH1 cells treated with 0.8% n-butanol and untreated cells at 0, 30, 80, and 195 minutes after addition. 3 biological replicates were grown, total RNA was extracted, labeled, and hybridized on 3 slides for each time point. http://www.microbesonline.org/cgi-bin/microarray/viewExp.cgi?expId=1266
Project description:Investigation of whole genome gene expression level changes in a E. coli fatty acid overproducing strain with or without heterologous expression of the M. luteus FabH. The strain expressing M. luteus FabH produces more methyl ketones. This study will be further described in Goh, E.B., E.E.K. Baidoo, J.D. Keasling, and H.R. Beller. Engineering of bacterial methyl ketone synthesis for biofuels. A 11 microarray study using total RNA recovered from six separate control cultures of Escherichia coli K-12 DH1 fatty acid overproducing strain with empty vector and five separate cultures of test strain, Escherichia coli K-12 DH1 fatty acid overproducing strain with vector overexpressing M. luteus FabH. Each chip measures the expression level of 4,254 genes from Escherichia coli K-12 with eight 60-mer probe pairs (PM/MM) per gene, with 2-fold technical redundancy.
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 6, 10, 14, and 26.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:The data explore the transcriptional response of strain LY180 and the furfural-resistant derivative EMFR9 to 0.5 g/L furfural LY180 and EMFR9 and differences in their expression profiles are described in Miller, E. N., L. R. Jarboe, L. P. Yomano, S. W. York, K. T. Shanmugam, and L. O. Ingram. 2009. Silencing of NADPH-dependent oxidoreductase genes (yqhD and dkgA) in furfural-resistant ethanologenic Escherichia coli. Appl Environ Microbiol 75:4315-23. The response of LY180 to furfural is described in Miller, E. N., L. R. Jarboe, P. C. Turner, P. Pharkya, L. P. Yomano, S. W. York, D. Nunn, K. T. Shanmugam, and L. O. Ingram. 2009. Furfural Inhibits Growth by Limiting Sulfur Assimilation in Ethanologenic Escherichia coli strain LY180. Appl Environ Microbiol., 2009. Total RNA was prepared from cultures of LY180 and EMFR9 immediately before and 15 min after addition of furfural to 0.5 g/L. The Nimblegen TI83333 chip measures expression of 4,237 genes, with 5 replicates, and 18 probes average per gene. Data from replicate 1 (OID 8641) and replicate 2 (OID 14117) was analyzed independently.
Project description:The data explore the transcription of strain LY180 and the yqhC deletion mutant LY180 del yqhC without and with 0.5 g/L furfural. LY180 and LY180 del yqhC are described in Turner, PC, EN Miller, LR Jarboe, P Pharkya, KT Shanmugam, and LO Ingram. 2009. Escherichia coli YqhC regulates transcription of the adjacent yqhD and dkgA genes, and mutations in yqhC contribute to furfural resistance in ethanologenic strains (in preparation for submission to Appl Env Microbiol) Total RNA was prepared from cultures of LY180 and LY180 del yqhC immediately before and 15 min after addition of furfural to 0.5 g/L. The Nimblegen TI83333 chip measures expression of 4,237 genes, with 5 replicates, and 18 probes average per gene.
Project description:Salmonella can infect a wide range of hosts and survive in the environment. This invasive pathogen has therefore evolved and acquired specific traits to cope with different, and in most cases unfavorable, conditions. In particular, transition metal ions are widely spread in these niches. These ions are essential in biology and play key roles in the structure-function of a large number of proteins. They can also be toxic, especially at high concentrations. Intracellular metal ion concentrations must therefore be tightly controlled to maintain normal metabolism. Salmonella has acquired traits to deal with the presence of toxic concentrations of some of these ions and, at the same time, to fulfill its requirement for essential metal ions when they are scarce. In this work we analyze the transcriptional response of Salmonella enterica to copper and zinc, two of these essential metals, in both rich (SLB) and defined (M9) media and at short times (10 minutes). This tiling-array based work provides a detailed description of the main transcriptional response when Salmonella detects fluctuations in copper and zinc concentrations. 14028s cells were grown up to OD 0.6, then, copper 10 uM (M9) or 1 mM (SLB) or Zn 50uM (M9) or 0.25 mM (SLB) was added and bacteria were grown for 10 minutes, RNA was isolated after this time. The RNA from bacteria grown without metal was also isolated. Each sample was analized by duplicate.
Project description:The data explore the transcriptional response of strains LY180 and EMFR9 to 5 mM furfural under anaerobic fermentation condition The data explore the transcriptional response of strains LY180 and EMFR35 to 15 mM furfural under anaerobic fermentation condition The expression differences of polyamine transporters in LY180 vs EMFR9 and EMFR35 are further described in RD Geddes,X Wang, LP Yomano, EN Miller, H Zheng, KT Shanmugam, and LO Ingram. 2013. Selected Polyamines and Polyamine Transporters Increase Furfural Tolerance (in preparation for submission to Appl Env Microbiol) Total RNA was prepared from cultures of LY180, EMFR9 and EMFR35 immediately before and 15 min after addition of furfural (5mM and 15 mM). The Nimblegen TI83333 chip measures expression of 4,237 genes, with 5 replicates, and 18 probes average per gene. The complete dataset comprising 8 samples is linked below as a supplementary file.