1Artie McFerrin Department of Chemical Engineering, Texas A&M University, College Station, 2Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Texas A&M University, College Station
We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.
Published January 27, 2014. Keywords: Bioengineering, split-intein, self-assembly, shear-thinning, enzyme, immobilization, organic synthesis
1Department of Biological Sciences, Center for Applied Chemical Biology, Youngstown State University
Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.
Published October 31, 2014. Keywords: Microbiology, Auxotroph, transposome, transposon, mutagenesis, replica plating, glucose minimal medium, complex medium, Enterobacter
1Department of Biochemistry, University of Toronto, 2Department of Molecular Genetics, University of Toronto, 3Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), University of Toronto
MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.
Published February 1, 2010. Keywords: Cellular Biology, protein-protein interaction, membrane, split-ubiquitin, yeast, library screening, Y2H, yeast two-hybrid, MYTH
JoVE Immunology and Infection
1Emory Vaccine Center at Yerkes National Primate Research Center, Emory University, 2Department of Pathology and Laboratory Medicine, Emory University
HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.
Published August 31, 2014. Keywords: Infectious Diseases, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
JoVE Immunology and Infection
1Department of Biological Sciences, Hunter College and Graduate Center, City University of New York, 2Sunnybrook Research Institute, Department of Immunology, University of Toronto
Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.
Published October 14, 2014. Keywords: Immunology, mouse, embryonic stem cells, in vitro differentiation, OP9 cells, Delta-like 1 (Dll-1) ligand, Notch, hematopoiesis, lymphocytes, T cells
1Scripps Florida, The Scripps Research Institute
Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.
Published June 20, 2014. Keywords: Chemistry, Split-and-pool synthesis, peptide tertiary amide, PTA, peptoid, high-throughput screening, combinatorial library, solid phase, triphosgene (BTC), one-bead one-compound, OBOC
1Institute for Molecular Biotechnology, RWTH Aachen University, 2Institute for Molecular Biology and Applied Ecology, Fraunhofer Gesellschaft
We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.
Published January 31, 2014. Keywords: Bioengineering, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
1Medical Research Council Centre for Regenerative Medicine, University of Edinburgh
This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.
Published October 26, 2011. Keywords: Developmental Biology, Stem Cells, hESC, Development, Endoderm, Liver, Hepatocyte, Endocrine Function, Exocrine Function
1Department of Surgery, Weill Cornell Medical College
A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.
Published February 25, 2013. Keywords: Stem Cell Biology, Molecular Biology, Cellular Biology, Medicine, Genetics, Developmental Biology, Anatomy, Surgery, Spermatogonial Stem cells, Stem cells, feeder cells, germ cells, testis, cell culture, microenvironment, stem cell niche, progenitor cells, mice, transgenic mice, animal model
1Division of Gastroenterology, Hepatology, and Nutrition, The Children's Hospital of Philadelphia, 2Department of Medicine, The Perelman School of Medicine at the University of Pennsylvania
A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.
Published June 5, 2014. Keywords: Medicine, Bile Ducts, Bile Ducts, Extrahepatic, Common Bile Duct, Bile Duct Diseases, Cell culture, bile duct, biliary atresia, Liver, gallbladder, fibrosis