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Gene Synthesis

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Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.
PLoS Genet. 2015 May;11(5):e1005005
Authors: Kast A,…

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Rosetta comparative modeling for library design: Engineering alternative inducer specificity in a transcription factor.

Proteins. 2015 May 13;

Authors: Jha RK, Chakraborti S, Kern TL, Fox DT, Strauss CE

Abstract
Structure-based rational mutagenesis for engineering protein functionality has been limited by the scarcity and difficulty of obtaining crystal structures of desired proteins. On other hand, when high throughput selection is possible, directed-evolution based approaches for gaining protein functionalities have been random and fortuitous with limited rationalization. We combine comparative modeling of dimer structures, ab initio loop reconstruction, and ligand docking to select positions for mutagenesis to create a library focused on the ligand contacting residues. The rationally reduced library requirement enabled conservative control of the substitutions by oligonucleotide synthesis and bounding its size within practical transformation efficiencies (∼10(7) variants). This rational approach was successfully applied on an inducer-binding domain of an Acinetobacter transcription factor, pobR, which shows high specificity for natural effector molecule, 4-hydroxy benzoate (4HB) but no native response to 3,4-dihydroxy benzoate (34DHB). Selection for mutants with high transcriptional induction by 34DHB was carried out at the single cell level under flow cytometry (via Green Fluorescent Protein expression under the control of pobR promoter). Critically, this selection protocol allows both selection for induction and rejection of constitutively active mutants. In addition to gain-of-function for 34DHB induction, the selected mutants also showed enhanced sensitivity and response for 4HB (native inducer) while no sensitivity was observed for a non-targeted but chemically similar molecule, 2-hydroxy benzoate (2HB). This is unique application of the Rosetta modeling protocols for library design to engineer a transcription factor. Our approach extends applicability of the Rosetta redesign protocol into regimes without a priori precision structural information. This article is protected by copyright. All rights reserved.

PMID: 25974100 [PubMed – as supplied by publisher]

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Correction: A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression.
PLoS One. 2015;10(5):e0126078
Authors: Hsiau TH, Sukovich D, Elms P, Prince RN, Strittmatter T, Rua…

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[Photogenerator of trichloroacetic acid–a perspective detritylation agent for microchip oligonucleotide synthesis].
Bioorg Khim. 2014 Sep-Oct;40(5):636-8
Authors:
Abstract
For the p…

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Differential gene expression analysis in early and late erythroid progenitor cells in β-thalassaemia.

Br J Haematol. 2015 Apr 19;

Authors: Forster L, McCooke J, Bellgard M, Joske D, Finlayson J, Ghassemifar R

Abstract
β- thalassaemia is a disorder of globin gene synthesis resulting in reduced or absent production of the β-globin chain in red blood cells. In this study, haematopoietic stem cells were isolated from the peripheral blood of six transfusion dependent β-thalassaemia patients and six healthy controls. Following 7 and 14 d in culture, early- and late- erythroblasts were isolated and purified. No morphological difference in maturation was observed following 7 d in culture, while a delayed maturation was observed in the patient group after 14 d. Following RNA isolation and linear amplification, gene expression analyses were performed using microarray technology. The generated data were analysed by two methods: the BRB-ArrayTools platform and the Bioconductor platform using bead level data. Following 7 d culture, there was no difference in gene expression between the control and patient groups. Following 14 d culture, 384 differentially expressed genes were identified by either analysis. A subset of 90 genes was selected and the results were confirmed by Quantitative-Real-Time-polymerase chain reaction. Pathways shown to be significantly altered in the patient group include apoptosis, MAPKinase and the nuclear factor-κB pathway.

PMID: 25892530 [PubMed – as supplied by publisher]

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A method for multiplex gene synthesis employing error correction based on expression.
PLoS One. 2015;10(3):e0119927
Authors: Hsiau TH, Sukovich D, Elms P, Prince RN, Stritmatter T, Ruan P, Curry B, Anderson P, Sam…

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Active site and laminarin binding in glycoside hydrolase family 55.

J Biol Chem. 2015 Mar 9;

Authors: Bianchetti CM, Takasuka TE, Deutsch S, Udell HS, Yik EJ, Bergeman LF, Fox BG

Abstract
The Carbohydrate Active Enzyme (CaZY) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β -1,3-glucanases. The founding structure of the GH55 is PcLam55A from the white-rot fungus Phanaerochaete chrysosporium (Ishida, T., et al. (2009) J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies implicate Glu502 as the catalytic acid (as proposed earlier for Glu663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the non-redundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities, and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate GH phylogenetic space for functional properties.

PMID: 25752603 [PubMed – as supplied by publisher]

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Duplex DNA and DNA-RNA Hybrids with Parallel Strand Orientation: 2′-Deoxy-2′-fluoroisocytidine, 2′-Deoxy-2′-fluoroisoguanosine, and Canonical Nucleosides with 2′-Fluoro Substituents Cause Unexpected Changes on the Double Helix Stability.

J Org Chem. 2015 Mar 5;

Authors: Ingale SA, Leonard P, Tran QN, Seela F

Abstract
Oligonucleotides with parallel or antiparallel strand orientation incorporating 2′-fluorinated 2′-deoxyribonucleosides with canonical nucleobases or 2′-deoxy-2′-fluoroisocytidine ((F)iCd, 1c) and 2′-deoxy-2′-fluoroisoguanosine ((F)iGd, 3c) were synthesized. To this end, the nucleosides 1c and 3c as well as the phosphoramidite building blocks 19 and 23 were prepared and employed in solid-phase oligonucleotide synthesis. Unexpectedly, (F)iCd is not stable during oligonucleotide deprotection (55 °C, aq NH3) and was converted to a cyclonucleoside (14). Side product formation was circumvented when oligonucleotides were deprotected under mild conditions (aq ammonia-EtOH, rt). Oligonucleotides containing 2′-fluoro substituents ((F)iCd, (F)iGd and fluorinated canonical 2′-deoxyribonucleosides) stabilize double-stranded DNA, RNA, and DNA-RNA hybrids with antiparallel strand orientation. Unexpected strong stability changes are observed for oligonucleotide duplexes with parallel chains. While fluorinated oligonucleotides form moderately stable parallel stranded duplexes with complementary DNA, they do not form stable hybrids with RNA. Furthermore, oligoribonucleotide duplexes with parallel strand orientation are extremely unstable. It is anticipated that nucleic acids with parallel chains might be too rigid to accept sugar residues in the N-conformation as observed for ribonucleosides or 2′-deoxy-2′-fluororibonucleosides. These observations might explain why nature has evolved the principle of antiparallel chain orientation and has not used the parallel chain alignment.

PMID: 25742047 [PubMed – as supplied by publisher]

Continue reading about Duplex DNA and DNA-RNA Hybrids with Parallel Strand Orientation: 2′-Deoxy-2′-fluoroisocytidine, 2′-Deoxy-2′-fluoroisoguanosine, and Canonical Nucleosides with 2′-Fluoro Substituents Cause Unexpected Changes on the Double Helix Stability.

Practical Synthesis of Cytidine-5-Carboxamide-Modified Nucleotide Reagents.
Nucleosides Nucleotides Nucleic Acids. 2015 Mar 4;34(3):180-198
Authors: Rohloff JC, Fowler C, Ream B, Carter JD, Wardle G, Fitzwater T

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Improved Lower Bounds of DNA Tags Based on a Modified Genetic Algorithm.
PLoS One. 2015;10(2):e0110640
Authors: Wang B, Wei X, Dong J, Zhang Q
Abstract
The well-known massively paral…

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