SOPs

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Method for analysis of various organic acids in the medium

Creator: Martijn Bekker

Contributor: The JERM Harvester

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Assay methodologies for individual glycolytic isoenzymes from the Mendes Group, University of Manchester, UK

Creator: Hanan Messiha

Contributor: Walter Glaser

Protocol for transfer of plasmids into Clostridium
acetobutylicum ATCC 824 by electroporation

Creators: None

Contributor: Ying Zhang

Purpose: Cation content analysis - protocol

Creator: Clara Navarrete

Contributor: The JERM Harvester

No description specified

Cloning and heterologous expression of gluconeogenic enzymes from S. solfataricus in E. coli

A refined and streamlined procedure to generate mutant in a wide range of different clostridial species, using group II intron retargeting methodologies.

Creators: Ying Zhang, Nigel Minton

Contributor: Ying Zhang

No description specified

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

This method describes how to derivatize the N-glutathionylspermidine and trypanothione produced by T. brucei trypanothione synthetase under in vivo-like conditions

Sample preparation - SOP for sampling, preparation of cell-free extracts, and determination of total extracted protein

Creator: Femke Mensonides

Contributor: Maksim Zakhartsev

The phosphorylation level of a particular protein can be determined using a procedure based upon western immunoblotting, with Phos-tag™ reagent present in the SDS-PAGE gel. The Phos-tag™ reagent, supplied in the form of Phos-tag™ acrylamide (Wako Pure Chemical Industries, AAL-107), causes proteins to be resolved both on the basis of size and phosphorylation state. This means that phosphorylated and de-phosphorylated forms of the same protein can be distinguished.

General protocol for measuring the kinetic parameters of the purified glycolytic enzymes from Saccharomyces cerevisiae - SOP for measuring the kinetic parameters of the purified glycolytic isoenzymes

Creator: Hanan Messiha

Contributor: Maksim Zakhartsev

No description specified

Experimental system that is designed to observe the in vivo stability and aggregation of a protein of interest.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

The procedure describes the preparation of fluorescent DNA probes from human mRNA or total
RNA.

Creator: Olga Krebs

Contributor: Olga Krebs

A protocol to improve conventional, recombination-based gene knock-out methodologies thtough the provision of negative selection markers, pyrE or codA.

Creators: Ying Zhang, Nigel Minton

Contributor: Ying Zhang

The Gene-doctoring method of lambda-red deletion (Lee et al., 2009) was modified slightly to create chromosomal mutations of fnr.

Creator: Matthew Rolfe

Contributor: Matthew Rolfe

Generating gene knock-ins using MJF618 expressing the defective lambdoid prophage recombination system λ red.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Generating gene knock-outs using DY330 expressing the defective lambdoid prophage recombination system λ red.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Protocol for transfer of plasmids into
Clostridium spp. by conjugation

Creators: Ying Zhang, Nigel Minton

Contributor: Ying Zhang

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

This is the general protocol for the glycolytic enzyme measurements. Detailed Information for each Enzyme can be found in the SOP: Assays for measuring the activities of the individual glycolytic isoenzymes of Saccharomyces cerevisiae

Creator: Hanan Messiha

Contributor: Walter Glaser

We routinely select specific RNAi gene targets (400–600 bp) and primers using the RNAit software http://trypanofan.path.cam.ac.uk/software/RNAit.html. A single pair of PCR primers are designed that incorporate four selected restriction sites (not present in the RNAi target fragment) such that a single PCR product can be differentially digested and sequentially cloned. For example, using MCS1/2, the following primers could be used to clone antisense followed by sense fragments: Primer 1, XbaI–BamHI-5′
...

No description specified
No description specified
No description specified

Labelling and extraction procedure for uniformly 13C-labelled E. coli (MG1655) for absolute quantification using isotope dilution technique by LC-MS

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Perturbation of starved cells with glucose. Concentrations of intra- and extracellular metabolites are followed in time.

Creator: Martijn Bekker

Contributor: Martijn Bekker

Protocol for applying a glucose perturbation in Streptococcus pyogenes.

Creator: Martijn Bekker

Contributor: Martijn Bekker

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.

Written Standard Operating Procedures provide workers with the operational
information necessary to perform a job properly and ensure consistency in the
operations. Standard Operating Procedures provide a historical record of steps in
the how, why and when and serve as a training tool for teaching users.

Creator: Olga Krebs

Contributor: Olga Krebs

This method describes the preparation of the in vivo-like buffer for the measurement of bloodstream T. brucei recombinant enzymes under pseudo-physiological conditions.

No description specified

Creator: Federico Rojas

Contributor: Federico Rojas

This protocol is designed to prepare sufficient amounts of high-quality total
RNA from the yeast saccharomyces cerevisiae grown in liquid culture for
analysis on microarrays.

Creators: Walter Glaser, Christa Gregori

Contributor: Walter Glaser

Method for synthesis of LAB-medium sued for the SYSMO-LAB project

Creator: Martijn Bekker

Contributor: The JERM Harvester

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

No description specified

Creator: Tomas Fiedler

Contributor: Tomas Fiedler

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

SOP for extracellular metabolite measurment

Purpose: PROTEIN EXTRACTION FOR 2-DE BASED PROTEOMIC EXPERIMENTS. 1. Citosoluble proteins

Creator: Miguel Curto Rubio

Contributor: The JERM Harvester

Purpose: PROTEIN EXTRACTION FOR 2-DE BASED PROTEOMIC EXPERIMENTS. 2. Membrane proteins

Creator: Miguel Curto Rubio

Contributor: The JERM Harvester

Method for transformation of plasmids into Lactococcus lactis

Creator: Martijn Bekker

Contributor: The JERM Harvester

Purpose:

Creator: Jose Ramos

Contributor: The JERM Harvester

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

Preparation of cell free extracts of the recombinant E. coli strains expressing the gluconeogenic S. solfataricus enzymes.

No description specified
No description specified

Creators: Louise Thomas, Maggie Smith

Contributor: Jay Moore

Purification of gluconeogenic enzymes from S. solfataricus in recombinant E.coli extracts

No description specified

Creators: Louise Thomas, Maggie Smith

Contributor: Jay Moore

This protocol for applying glucose perturbations works for Lactococcus lactis and Enterococcus faecalis

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

This assay uses a dual-wavelength spectrophotometer to quantify cytochromes present in the E. coli respiratory chain.

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

This method describes how one can quench metabolism of Escherichia coli and extract metabolites from many kinds of metabolite classes like: nucleotides, sugar-phosphates, organic acids ....

Method extraction of intracellular metabolites in Lactococcus lactis

Creator: Martijn Bekker

Contributor: The JERM Harvester

A protocol for acidic quenching of lactic acid bacteria used for analyses of intracellular metabolites.

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.

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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.

No description specified

Creator: Holger Janssen

Contributor: The JERM Harvester

Method extraction of intracellular metabolites in Lactococcus lactis

No description specified
No description specified

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.

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

This protocol describes the transcriptional profiling of E. coli cultures using microarrays. The protocol utilises RNA isolated as described in another SOP (SUMO RNA isolation from E. coli) and with hybridisation to Ocimum Ocichip E. coli K-12 microarrays.

For the study of mRNA decay rates, transcription was inhibited with ActinomycinD, and RNA splicing with Sinefungin, at different time points, in the Matthews lab. rRNA depleted RNA was extracted from each of the samples in the Clayton lab, and sent for deep sequencing at the BioQuant facility in Heidelberg

Sample preparation procedure for metabolic footprint analysis on T. b. brucei 427 using LC/MS

No description specified
No description specified

Creator: Michael Kohlstedt

Contributor: Michael Kohlstedt

This is a well-established, classical genetic method of constructing chromosomal monolysogenic fusions to a promoterless lacZ gene.

ClosTron mutants should always be subjected to Southern blot analysis to ensure that only
one intron insertion has occurred.

Creators: Ying Zhang, Nigel Minton

Contributor: Ying Zhang

No description specified

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

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.

No description specified

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

No description specified

Creators: Louise Thomas, Maggie Smith

Contributor: Jay Moore

No description specified

Creators: Louise Thomas, Maggie Smith

Contributor: Jay Moore

No description specified

Creators: Louise Thomas, Maggie Smith

Contributor: Jay Moore

Isolation of total RNA from Bacillus Subtilis using phenol-chloroform extraction method by maintaining cryogenec conditions initailly to prevent RNA degradation. Quality of the obtained RNA is then tested with Agilent Bioanalyser before proceeding for gene expression analysis.

An overview of creating MAGE-TAB compliant spreadsheets for transcriptomics data in SysMO SEEK

This is a protocol for determining the activity of the T. brucei Trypanothione synthetase under in vivo-like conditions.

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

Creator: Michael Ederer

Contributor: Michael Ederer

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

Creator: Lisbeth Lyngberg

Contributor: Lisbeth Lyngberg

No description specified
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