Farnesyl Diphosphate (FPP) Toxicity in E. coli, M9 medium
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ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Robert Dahl
PROVIDER: E-GEOD-30403 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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.
2011-05-13 | E-GEOD-29267 | biostudies-arrayexpress
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.
2011-07-07 | GSE30403 | GEO
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.
2011-05-13 | GSE29267 | GEO
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
2010-05-16 | E-GEOD-16973 | biostudies-arrayexpress
Project description:Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many Gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences. We used microarrays to profile the transcriptional changes incurred when downregulating mvaS, mvaA, or mvaK in S. aureus. Keywords: stress response S. aureus mutants were generated in which mvaS, mvaA, or mvaK were placed under control of the IPTG-inducible Pspac promoter. These mutants, as well as wildtype RN4220 S. aureus, were grown with or without 1 mM IPTG, harvested in exponential phase, and their total RNA was extracted and hybridized to Affymetrix GeneChips. Experiments were done in triplicate.
2010-05-05 | E-GEOD-13424 | biostudies-arrayexpress
Project description:In this work expression of two rhsA elements, ext-a1 and daORF-a1, was differentially induced using IPTG. RhsA is a member of multigene rhs family and can code for five different open reading frames (ORFs), referred to as genetic elements. The functions of rhsA and the different ORFs it codes are not well understood. We used microarrays to study the effect of over-expression of two rhsA elements, ext-a1 and daORF-a1, on E. coli cells. Keywords: time course E. coli cells were harvested from biological replicate cultures at 0 hrs (0 mM IPTG) and at 1.5 and 3.5 hrs (0, 0.1, and 1 mM IPTG) after IPTG induction for RNA extraction and hybridization on Affymetrix arrays. A total of seven different conditions were analysed. Two biological replicates and two technical replicates were performed for each condition (a total of 28 microarray hybridizations).
2011-05-15 | E-GEOD-12411 | biostudies-arrayexpress
Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data.
2009-08-07 | GSE17505 | GEO
Project description:The transcriptome profiles for wild-type (plasmid-free) and recombinant (plasmid-bearing) Escherichia coli during well-controlled synchronized high-cell-density fed-batch cultures were analyzed by DNA microarrays. It was observed that the growth phase significantly affected the transcriptome profiles, and the transcriptome profiles were significantly different for the recombinant and wild-type cultures. The response of the wild-type and recombinant cultures to an isopropyl-1-thio-β-D-galactopyranoside- (IPTG-) addition was examined, where IPTG induced recombinant protein production in the plasmid-bearing cultures. The IPTG-addition significantly altered the transcriptome response of the wild-type cultures entering the stationary phase. The IPTG-induced recombinant protein production resulted in a significant down-regulation of many energy synthesis genes (atp, nuo, cyo), as well as nearly all transcription- and translation-related genes (rpo, rpl, rpm, rps, rrf, rrl, rrs). Numerous phage (psp, hfl) and transposon-related genes (tra, ins) were significantly regulated in the recombinant cultures due to the IPTG-induction. These results indicate that the signaling mechanism, associated with the recombinant protein production, may induce a metabolic burden in the form of a phage defense mechanism. Taken together, these results indicated that recombinant protein production initiated a cascade of transcriptome responses that down-regulated the very genes needed to sustain productivity. In this study, the transcriptome profiles for recombinant and wild-type E. coli cultures were compared. The recombinant and wild-type cultures were monitored during the exponential growth phase and as the cultures entered the stationary phase. Well-controlled fed-batch fermenters were used to synchronize the high-cell density cultures. E. coli MG1655 [pPROEx-CAT] and E. coli MG1655 (plasmid-free) were exposed to IPTG in the exponential phase, where chloramphenicol acetyltransferase (CAT) expression was induced in the plasmid-bearing cultures. Control cultures, not exposed to IPTG, were also examined. The gene expression profiles were determined using DNA microarrays. Recombinant protein production was shown to result in a metabolic burden due to significant down-regulation of the transcription, translation, and energy synthesis genes. This was the first comprehensive study that has examined the transcriptome of wild-type and recombinant cultures under the same set of conditions. This data indicated that one cannot merely extrapolate the behavior of recombinant cultures from wild-type culture data. Experiment Overall Design: All fermentations were synchronized in time to the time at which the cell density reached approximately 11.5 OD, or roughly 70% of the fermenter’s maximum cell density. IPTG (5 mM) was added when the cell density reached the targeted OD for the cultures to be induced. Cells were harvested at 0, 1, 2, 3, 4, and 5 hours relative to the synchronization time point (identified as Time S0, S1, etc). Each fermentation condition was conducted in duplicate. For the recombinant Control Time S0 sample, triplicate samples were available since prior to the IPTG-addition, all fermentations within a strain were replicates. All fermentation conditions were repeated twice (two biological replicates). RNA from each biological replicate was purified and processed independently. Three biological replicates were obtained from for the recombinant Control sample (Time S0) since the cultures prior to the IPTG-addition were replicates. Prior to hybridization, where only two biological replicates existed, one of the RNA samples was divided into two technical replicates, resulting in three separate hybridized chips. The wild-type Time S0 samples and all Time S1 and S4 samples contained data from three DNA microarrays obtained from two biological duplicates (30 DNA microarrays total).
2009-08-25 | E-GEOD-17505 | biostudies-arrayexpress
Project description:gene expression profiles by overexpressing Hha from pCA24N-hha using 2 mM IPTG induction in BW25113 wild type suspension cells in LB medium at 37C relative to BW25113 carrying the empty vector plasmid in the same test conditions. Experiment Overall Design: Strains: E. coli K12 BW25113/pCA24N, BW25113/pCA24N-hha Experiment Overall Design: Growth conditions: grow culture in LB medium at 37C to OD600 0.1 and add 2 mM IPTG to induce, and grow to OD600 0.5 to collect cells. RNA was extraction, and gene expression profiles were evaluated.
2008-10-20 | E-GEOD-10116 | biostudies-arrayexpress
Project description:This experiment was conducted to understand the gene functions of Escherichia coli in different concentrations of induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Experiments were performed with no induction, or with induction using 0.2mM or 1 mM of IPTG.
2022-06-09 | E-MTAB-11808 | biostudies-arrayexpress