SOPs

SOPs related with medium composition, fermentation and stocking of S. solfataricus.

MIAPE (Minimum Information About a Proteomics Experiment) is the recommended format for proteomics data in SysMO-SEEK. The document attached provides more information and links to tools and resources.

Standard procedure regarding intracellular metabolome analysis using GC-MS.

Protocol for RNA isolation, cDNA synthesis and labeling and hybridization and cleanup of Sulfolobus solfataricus microarray

Standard operating procedures regarding iTraq based proteomics.

A model to describe the effect of K+ uptake by KdpFABC on the two-component system KdpD/KdpE, which enables the identification of control principles of the Kdp system

The reverse transcriptase synthesizes DNA, which complements the mRNA template (complementary DNA, cDNA). Cy3/Cy5-dCTP are incorporated into cDNA during Reverse transcription. The obtained Cy3/Cy5 cDNA are then competitively hybridised onto Agilent microarray slide and subsequently scanned.

Cells were harvested from culture keeping the cells cold to quench the physiological condition of RNA and the cells were mechanically disrupted. RNA was isolated from the cells by conventional acid-phenol method and the quality was checked by Agilent bioanalyser.

This SOP describes the SUMO procedure for determining B-galactosidase activities.

A kinetic model that describess the activation of a dimeric efflux system that could bind either GSH or SLG

This SOP defines the format of SOPs used in the SUMO consortium.

A model for translation elongation, which allows the prediction of how different conditions and parameters affect the rate and throughput of translation.

A model to describe aggregation of homomeric protein complexes in mechanosensitive channels in E.coli.

Measurement of the transmembrane pH gradient and thus pHi, when the external pH is known.

A procedure to analyse the genomic interaction of E.coli RNA polymerase (RNAP) upon methylglyoxal (MG) stress, a toxic keto-aldehyde by-product of metabolism.

SOP used for detecting non influential parameters and interactions in non linear dynamical models. These parameters can be estabilished which allows the prioritization of parameters that can be subsequently estimated using robust global optimizations methods.

The model describes the series of chemical reactions in MG detoxification pathway, allowing one to predict the flux of all compounds produced during detoxification.

Mathematical model of pH buffering, which allows the prediction of how the buffering capacity depends on the cytoplasm's composition, for any number of buffers.

Method of how to perform the purification of glyoxalase II as well as kinetics assays.

A method of how to measure methylglyoxal present in the growth medium.

Measurements of K+ retained in the cytoplasm using flame photometry.

The fluorescent DNA-binding dye used in qRT-PCR binds to all kinds of double stranded DNA. To prevent false-positive results, the RNA is treated with DNase to remove remaining DNA.

Quantitative real-time PRC is used to compare kdpFABC expression between the E. coli strains MG1655 (wildtype)and MG1655 (kdpA4, a kdpFABC-inactive mutant after a shift to K+ limitation.

A method to compare kdpFABC expression between MG1655 (wildtype) and MG1655 (kdpA4) after a shift to K+ limitation, the RNA was extracted from samples taken at different points in time.

Pulsed-FRAP measure the diffusion constants in confined volumes in small cells like E. coli and other bacteria or cellular organelles.

A procedure to measure the levels of GSH and SLG before and after exposure to MG using formic acid. A similar expreimental set up as for potassium efflux experiments is used to which a silicon oil centrifugation step is incorporated. Samples are analysed by LC-MS-MS in order to quantify intracellular GSH and SLG levels.

The reverse transcriptase synthesizes DNA, which complements the mRNA template.

To induce kdpFABC expression, cells are cultivated in K10 minimal medium (10mM K+) were shifted into K+ limiting growth medium (K0), containing 20 μM K+ by filtration.

FRAP experiments are used for studying cytoplasmic diffusion in cells and cells membrane.

Growing Escherichia coli to express KefF by adding IPTG for purification and kinetics experiments.