Position effect on secretion efficiency v1

Mapping Intimacies ◽

10.17504/protocols.io.b3t9qnr6 ◽

2022 ◽

Author(s):

Joao Vitor Molino

Keyword(s):

Relative Position ◽

Expression Vector ◽

Position Effect ◽

Secretion Efficiency

A proposed method to evaluate secretion efficiency dependency on relative position inside the expression vector.

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Adaptation of the Highly Productive T7 Expression System to Streptomyces lividans

Applied and Environmental Microbiology ◽

10.1128/aem.02186-09 ◽

2009 ◽

Vol 76 (3) ◽

pp. 967-970 ◽

Cited By ~ 21

Author(s):

François-Xavier Lussier ◽

François Denis ◽

François Shareck

Keyword(s):

Expression Vector ◽

Expression System ◽

Streptomyces Lividans ◽

T7 Rna Polymerase ◽

Polymerase Gene ◽

Positive Bacterium ◽

Secretion Efficiency ◽

Extracellular Protease Activity ◽

T7 Expression System ◽

Rna Polymerase Gene

ABSTRACT Streptomyces lividans is a Gram-positive bacterium known for its remarkable secretion efficiency and low extracellular protease activity. In the present work, we adapted the highly productive T7 expression system to S. lividans. A codon-optimized T7 RNA polymerase gene was chromosomally integrated, and a bifunctional T7 expression vector was constructed.

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Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180161 ◽

1990 ◽

Vol 48 (1) ◽

pp. 282-283

Author(s):

José L. Carrascosa ◽

José M. Valpuesta ◽

Hisao Fujisawa

Keyword(s):

Dna Sequences ◽

Expression Vector ◽

Three Dimensional ◽

Deae Cellulose ◽

Dna Packaging ◽

Dimensional Structure ◽

Two Dimensional ◽

Freezing And Thawing ◽

Three Dimensional Structure ◽

Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

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