Wetlab approach to transcription fidelity

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Bistable switches are the key elements of the regulatory networks governing cell development, differentiation and life-strategy decisions. Transcriptional noise is a main determinant that causes switching between different states in bistable systems. By using the human pathogen Streptococcus pneumoniae as a model bacterium, we will investigate how transcriptional fidelity and processivity influence (noisy) gene expression and participate in bistability. To study this question, we will use both ...

For cells to accurately read out the genomic content, high fidelity during transcription is required. This is mainly established by the accuracy of the active centre of RNA polymerase (RNAP). Based on in vitro experiments with Escherichia coli RNAP it was also suggested that proofreading of transcription via RNA hydrolysis by RNAP may contribute to overall fidelity and processivity. RNAP’s intrinsic cleavage activity is stimulated by the highly conserved Gre factors suggesting that Gre factors ...

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard ...

Here we develop a set of new tools for S. pneumoniae and as a case study we show that S. pneumoniaea SMC is recruited to oriC by ParB and promotes chromosome segregation.

Cells were grown to mid-exponential phase (OD600nm ~0.2) in GM17 medium at 37°C (with 0.15 mM ZnSO4 where relevant) and 84 ml of culture was mixed by inverting with 8.4 ml of fixing solution (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EGTA, 1 mM EDTA, 30% (v/v) formaldehyde) and incubated at room temperature for 30 min. Cells were disrupted and crosslinked DNA was sheared by sonication. Antibodies coupled to magnetic beads were used to pull down cross-linked complexes. DNA was purified, amplified, ...

Artificial transcription elongation compexes are assembled in vitro using synthetic deoxy-oligonucleotides (representing template and non template DNA strands), radiolabelled RNA (representing nascent transcript) and purified RNA polymerase. After high salt wash the incorrect NTP is added and reaction is allowed to proceed for the various amounts of time. Reaction is stopped by addition of formamide-containing loading solution and the products are resolved on high-percentage denaturing polyacryamide ...

S.pneumoniae D39 cells (wild type and delta greA) were grown in C+Y medium and cells were harvested for total RNA isolation at mid-exponential growth (OD600nm 0.3 for wt, 0.25 for delta greA). Total RNA was isolated as described before (Kloosterman et al 2006). The total RNA samples were examined by capillary electrophoresis. dephosphorylated with antarctic phosphatase followed by treatment with polynucleotide kinase (PNK). Afterwards, samples were poly(A)-tailed using poly(A) polymerase. Then a ...

1. Preparation of B. subtilis cultures

Inoculate cells from -80°C stocks in 10 ml time-lapse microscopy (TLM) medium (62 mM K2HPO4 , 44mM KH2PO4, 15 mM (NH4)2SO4, 6.5 mM sodium citrate, 0.8 mM MgSO4, 0.02 % casamino acids, 27.8 mM glucose, 0.1 mM L-tryptophan, the pH was set to 7 using a KOH solution) supplemented with antibiotics, if necessary. Grow the cells overnight in a shake flask (30°C, 225 rpm). The following morning, dilute the cells 1:10 in pre-warmed chemically defined medium (CDM) (62 ...

WT 110901_SN865_B_L006_R1_GQC-28- ATCACG.fastq.gz 100 19.624.852 1,29

llumina fastq format 4 lines for each sequence: 1- Unique identifier, with the following format: @::::#/ 2- Sequence (A, T, C ,G or N (undetermined) only) 3- Orientation (always forward without mapping) 4- Quality value for each base, corresponding to a Phred-like score encoded in ASCII format, with an offset of of 33 (e.g. “J” gives a value of 41) and is in accordance with sanger FASTQ format. The sequence file is compressed ...

See wild type sample.

GEO format data