Transcription profiling of G sulfurreducens GGS Fe(III) vs fumarate
ABSTRACT: G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, (see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press. for more information), with acetate (5 mM) as the electron donor and Fe(III) citrate (55 mM) or fumarate (27.5 mM) as the electron acceptor. Under these conditions acetate is the substrate limiting growth. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at 80 °C prior to RNA extraction. Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of acetate limited growth with Fe(III) as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.
A DNA microarray representing the genome of Geobacter sulfurreducens was constructed for use in global gene expression profiling of cells under steady-state conditions with acetate as the electron donor and Fe(III) or fumarate as the electron acceptor. Reproducible differences in transcript levels were also observed in comparisons between cells grown with ammonia and those fixing atmospheric nitrogen. There was a high correlation between changes in transcript levels determined with microarray an ...[more]
Project description:G. sulfurreducens wild type and pilR mutant (GSU1495) were grown in chemostats for RNA extraction used for microarray analysis and qRT-PCR. The electron donor (acetate 5mM) was limiting at a dilution rate of 0.05 h-1 and ferric citrate(55mM) was used as the electron acceptor at 30C, as previously described (Esteve-Nunez et al., 2005). Analysis of acetate, Fe(II) and protein were performed as previously described (Esteve-Nunez et al., 2005). Disruption of the pilR gene (GSU1495) was made in G. sulfurreducens strain DL1 (ATCC 51573) by the recombinant PCR and single-step recombination method (Murphy et al., 2000), essentially as described (Lloyd et al., 2003). To disrupt the pilR gene a 2.25 kb DNA fragment was constructed by PCR in which 0.39 kb of the pilR coding sequence (codons 192 - 323) were replaced with the kanamycin resistance cassette (Knr) of pBBR1MCS-2 (Kovach et al., 1995). This fragment consisted of 29 bp of upstream sequence together with the first 575 bp of the pilR gene, followed by the kanr cassette (1.1 kb), and the last 409 bp of the pilR gene and 132 bp of downstream sequence. The mutant was selected in NBAF plates supplemented with kanamycin and incubated at 30C in an anaerobic chamber containing a mixture of 7% H2, 10% CO2, 83% N2. A single kanamycin-resistant colony was selected, tested for the insertion of the Knr cassette by PCR, and designated DLJK3.
Project description:Exponentially growing Sulfolobus acidocaldarius were treated with NaAc to generate replication runout and arrest in G2 phase. The cells were then resuspended in fresh acetate-free media which generates a synchronous population. Samples for investigation of gene expression change were taken during the synchronised populations progress through the cell cycle.
Project description:We investigated the effect that intragenic s54 binding sites have on transcripton elongation. In an rpoN deletion we have over-expressed rpoN from a plasmid (with vector only contol) and then looked at changes in transcription elongation around intragenic s54 binding sites by RNA-seq. We are also looking at changes in transcription initiation/elongation at sites of sigma factor overlap.
Project description:Mice were obtained from in house breeding of C57BL/6J and C57BL/6J-Chr 1A/Na breeding pairs (Jackson Laboratories, USA). To produce F1 hybrids, C57BL/6J females were mated with C57BL/6J-Chr 1A/Na males. The F1 hybrids were intercrossed, producing 82 F2 progeny (41 males and 41 females). Microarray analysis was performed on six pairs of affected and non-affected male animals from the F2 progeny selected on the basis of their motor activity levels (average daily levels of distance moved over a 3 days recording: 768±74 cm/hr (affected) versus 1765±175 cm/hr (non-affected)(p<0.0001).
Project description:We measured global RNA levels using RNA-seq in cas3+ E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect RNA levels.
Project description:Using RNAseq, the goal of the study is to investigate transcription factor regulation of virulence genes in 14028s Salmonella Typhimurium grown in SPI-1-inducing conditions. Transcription factors were cloned into pBAD24. RNA levels were compared in 14028s Salmonella Typhimurium with the transcription factor-encoding gene deleted, carrying either empty pBAD24, or pBAD24 expressing the transcription factor corresponding to the deletion. The Transcription factors analyzed are HilA, HilC, HilD, SprB, InvF, RtsA and RtsB.
Project description:We measured global RNA levels using RNA-seq in cas3 E. coli cells with intact CRISPR arrays or cells with the CRISPR-I array deleted to determine if off-target events driven by endogenous spacers affect local gene expression.
Project description:All yeast strains were in the genetic background W303-1A (MATa leu2-3,112 trp1-1 ura3-1 can1-100 ade2-1 his3-11,15). Gene expression profiles of the following mutant strains were compared with WT: YIA29 (mdl1::HIS3); YIA30 (yme1::KAN); YIA31 (mdl1::HIS3 yme1::KAN); 2 independent RNA preparations per strain were used (A and B); 2 independent array hybridisations per RNA preparation were performed (color switch experiments).