SOPs

Method extraction of intracellular metabolites in Lactococcus lactis

Method extraction of intracellular metabolites in Lactococcus lactis

Method for transformation of plasmids into Lactococcus lactis

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

Method for analysis of various organic acids in the medium

Protocol for applying a glucose perturbation in Streptococcus pyogenes.

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

This HPLC method uses a isocratic method and a RI detector to identify and quantify almost all excreted catabolic metabolites.

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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 ....

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

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

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

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

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

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

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

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

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

Protocol for transfer of plasmids into Clostridium spp. by conjugation

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

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.

This SOP describes the preparation of E. coli RNA

SOP for growing yeast in anaerobic conditions

Biomass sampling - SOP for sampling of biomass

Metabolic perturbations - SOP for metabolic perturbations (i.e. glucose pulse)

Yeast strains used in the project

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

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

Sample preparation - SOP for sampling, preparation of cell extracts, and general assay set-up

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′ ...

SOP for extracellular metabolite measurment

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.

The kit is used for the quantitative detection of ATP in yeast cells by luciferase driven bioluminescence. ATP extraction is done by boiling cells in TE buffer.

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.

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

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

Purpose: Cation content analysis - protocol

Purpose:

SOP for ß-Galactosidase assay.

SOP for shake flask cultivation of B.Subtilis in Bacell-Sysmo

This file describes how to isolate mRNA from E. coli using the kit from Epicentre, for gene expression analysis via RT-PCR

This document describes the chemostat conditions to run comparable experiments in different bioreactors in different labs.

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.

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

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

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