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Gene Synthesis

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Synthetic approach to the generation of antibody diversity.
BMB Rep. 2015 Jul 1;
Authors: Shim H
Abstract
The in vitro antibody discovery technologies revolutionized the generation o…

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Orf virus inhibits interferon stimulated gene expression and modulates the JAK/STAT signalling pathway.

Virus Res. 2015 Jun 22;

Authors: Harvey R, McCaughan C, Wise LM, Mercer AA, Fleming SB

Abstract
Interferons (IFNs) play a critical role as a first line of defence against viral infection. Activation of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway by IFNs leads to the production of IFN stimulated genes (ISGs) that block viral replication. The Parapoxvirus, Orf virus (ORFV) induces acute pustular skin lesions of sheep and goats and is transmissible to man. The virus replicates in keratinocytes that are the immune sentinels of skin. We investigated whether or not ORFV could block the expression of ISGs. The human gene GBP1 is stimulated exclusively by type II IFN while MxA is stimulated exclusively in response to type 1 IFNs. We found that GBP1 and MxA were strongly inhibited in ORFV infected HeLa cells stimulated with IFN-γ or IFN-α respectively. Furthermore we showed that ORFV inhibition of ISG expression was not affected by cells pretreated with adenosine N1-oxide (ANO), a molecule that inhibits poxvirus mRNA translation. This suggested that new viral gene synthesis was not required and that a virion structural protein was involved. We next investigated whether ORFV infection affected STAT1 phosphorylation in IFN-γ or IFN-α treated HeLa cells. We found that ORFV reduced the levels of phosphorylated STAT1 in a dose-dependent manner and was specific for Tyr701 but not Ser727. Treatment of cells with sodium vanadate suggested that a tyrosine phosphatase was responsible for dephosphorylating STAT1-p. ORFV encodes a factor, ORFV057, with homology to the vaccinia virus structural protein VH1 that impairs the JAK/STAT pathway by dephosphorylating STAT1. Our findings show that ORFV has the capability to block ISG expression and modulate the JAK/STAT signalling pathway.

PMID: 26113305 [PubMed – as supplied by publisher]

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Using Plasmids as DNA Vaccines for Infectious Diseases.
Microbiol Spectr. 2014 Dec;2(6)
Authors: Tregoning JS, Kinnear E
Abstract
DNA plasmids can be used to induce a protective (or …

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DNA Assembly Tools and Strategies for the Generation of Plasmids.
Microbiol Spectr. 2014 Oct;2(5)
Authors: Baek CH, Liss M, Clancy K, Chesnut J, Katzen F
Abstract
Since the discovery…

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Understanding how cis-regulatory function is encoded in DNA sequence using massively parallel reporter assays and designed sequences.
Genomics. 2015 Jun 10;
Authors: White MA
Abstract

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[Expression and characterization of Coprinus cinereus peroxidase].

Wei Sheng Wu Xue Bao. 2015 Mar 4;55(3):321-9

Authors: Dong B, Niu Q, Zhang W, Geng S, Li P, Yuan W, Gong Y, Liang K

Abstract
OBJECTIVE: The aim of our study is to express Coprinus cinereus peroxidase (CIP) in Pichia Pastori efficiently.
METHODS: We synthesized CIP gene with P. pastori codon bias by our Gene Synthesis and site-specific mutagenesis platform, using DNAWorks 3.1 program to design and optimize primers. Then, we sequenced the PCR products, inserted the correct gene into expression vector pPICZαA and transformed the linearized pPICZαA-Cip DNA into P. pastori GS115. We integrated CIP gene into the genome of P. pastori, using the α-mating factor from Sacchoramyces cerevisiae as signal peptide to direct the secretion of the recombinant protein. To obtain transformants with high CIP activity, we checked transformants by nested PCR and stained 82 positive ones on YPD agar plate with 1000 mg/L Zeocin. Then, we got 6 transforments with high resistance to Zeocin and expressed them in small scale; the one exhibiting the highest activity was chosen as engineered strain and named CIP/Gs115.
RESULTS: We purified CIP from culture medium after induction with ethanol, the maximum activity reached 487.5 U/mL on the 4th day. The purified CIP exhibited maximal activity at pH 5.0 and 25 degrees C with ABTS as substrate. The enzyme had 61.5% of the maximal activity at 45 degrees C and was stable below 40 degrees C. However, the stability was drastically reduced above 45 degrees C. The recombinant CIP remained stable between pH 4.5 and 6.5. We studied the substrate specificity on different substrates with the purified enzyme, and the optimal substrates were in the order of ABTS > 2, 6-Dimethoxyphenol > guaiacol > 2, 4-Dichlorophenol > phenol.
CONCLUSION: The highly secretory expression of CIP and high special activity lay the good foundation for it’ s industrial applications in waste water treatment, decolouration of dyestuffs.

PMID: 26065274 [PubMed – in process]

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RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli.
Sci Rep. 2015;5:11302
Authors: Zampini M, Stevens PR, Pachebat JA, Kingston-Smith A, Mur LA, Hayes F
Ab…

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Exploring codon context bias for synthetic gene design of a thermostable invertase in Escherichia coli.
Enzyme Microb Technol. 2015 Jul-Aug;75-76C:57-63
Authors: Pek HB, Klement M, Ang KS, Chung BK, Ow DS, Lee DY

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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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