1Department of Molecular Genetics, University of Toronto, 2Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, 3Department of Biochemistry, Research and Innovation Centre, University of Regina
Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.
Published November 12, 2012. Keywords: Genetics, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
1Children's Mercy Hospital and Clinics, School of Medicine, University of Missouri-Kansas City
This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.
Published February 13, 2013. Keywords: Genetics, Molecular Biology, Immunology, Medicine, Genomics, Proteins, RNA-seq, Next Generation DNA Sequencing, Transcriptome, Transcription, Thrombin, Endothelial cells, high-throughput, DNA, genomic DNA, RT-PCR, PCR
1Department of Biotechnology, Delft University of Technology, 2Delft Center for Systems and Control, Delft University of Technology
A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via β-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.
Published October 2, 2012. Keywords: Bioengineering, Microbiology, Biochemistry, Chemistry, Chemical Engineering, Oil remediation, alkane metabolism, alkane hydroxylase system, resting cell assay, prefoldin, Escherichia coli, synthetic biology, homologous interaction mapping, mathematical model, BioBrick, iGEM
1Department of Energy, Environmental and Chemical Engineering, Washington University, 2Department of Biology, Washington University, 3Department of Energy, Environmental and Chemical Engineering and Department of Biology, Washington University
13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.
Published January 26, 2012. Keywords: Molecular Biology, GC-MS, novel pathway, metabolism, labeling, phototrophic microorganism
1Molekulare Pflanzenphysiologie, Max-Planck-Institut
Combination of genomics, co-expression gene analysis and the identification of target compounds via metabolism give gene functional annotation.
Published June 17, 2012. Keywords: Plant Biology, Genetics, Bioinformatics, Metabolomics, Plant metabolism, Transcriptome analysis, Functional annotation, Computational biology, Plant biology, Theoretical biology, Spectroscopy and structural analysis
1Department of Biological Sciences, Florida Atlantic University, 2Department of Chemistry & Biochemistry, Florida Atlantic University
A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.
Published April 15, 2012. Keywords: Neuroscience, Drosophila melanogaster, Giant Fiber Circuit, screening, in vivo, nanoinjection, electrophysiology, modulatory compounds, biochemistry
1Institute of Healthy Ageing, and GEE, University College London - UCL, 2School of Biosciences, University of Kent
The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.
Published January 14, 2011. Keywords: Neuroscience, Drosophila melanogaster, electrophysiology, Giant Fiber System, flight muscles, nervous system
JoVE Immunology and Infection
1Department of Molecular and Biomedical Sciences, University of Maine, Orono, 2Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono
Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.
Published November 1, 2013. Keywords: Immunology, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
1Chemical and Biological Engineering Department, State University of New York at Buffalo, 2Chemical Engineering Department, Massachusetts Institute of Technology
The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.
Published January 13, 2013. Keywords: Biomedical Engineering, Chemical Engineering, Bioengineering, Molecular Biology, Cellular Biology, Microbiology, Basic Protocols, Biochemistry, Biotechnology, Heterologous biosynthesis, natural products, antibiotics, erythromycin A, metabolic engineering, E. coli
JoVE Clinical and Translational Medicine
1Tumor Microenvironment and Metastasis Program, Wistar Institute, 2Department of Pathology and Lab Medicine, Perelman School of Medicine, University of Pennsylvania, 3Department of Microbiology and Immunology and Department of Genetics, Geisel School of Medicine at Dartmouth, 4Division of Endocrine and Oncologic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, 5Rena Rowan Breast Center, Abramson Cancer Center, University of Pennsylvania, 6Center for Advanced Medicine, University of Pennsylvania
Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.
Published March 26, 2014. Keywords: Medicine, Transgenic mice, breast cancer, metastasis, intraductal injection, latent mutations, adenovirus-Cre