Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins in<i>E. Coli</i>and<i>Bacillus Megaterium</i>and Assessment of the GFP-Amylase Thermostability

Atanassov, Ivan; Stefanova, Katerina; Tomova, Iva; Kamburova, Margarita

doi:10.5504/bbeq.2013.0079

Cited by 3 publications

(1 citation statement)

References 28 publications

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“…Two pairs of specific primers (Supplemental Table 1S) were designed respectively according to the DNA sequences of the Cry218-pht304 plasmid and PHKT2 recipient expression vector [10]. These amplified DNA fragments were mixed at a ratio of 1:3 (100 ng vector to 300 ng DNA fragments), and transformed into E. coli XL-10 Gold cells [11]. The recombinant plasmid was DNA sequenced to confirm the construction by TakaRa (Dalian, China).…”

Section: Generation Of Recombinant Plasmidsmentioning

confidence: 99%

Engineering the bacterial endophyte Burkholderia pyrrocinia JK-SH007 for the control of lepidoptera larvae by introducing the cry218 genes of Bacillus thuringiensis

Yang

Zhong

et al. 2017

Biotechnology & Biotechnological Equipment

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To expand the usage of endophytes in agriculture and in forestry, the insecticidal gene cry218 of Bacillus thuringiensis was introduced into a poplar bacterial endophyte Burkholderia pyrrocinia JK-SH007. The cry218 gene was cloned by polymerase chain reaction (PCR) and was inserted into a PHKT 2 expression vector that was introduced into the bacterial endophyte JK-SH007. By using sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and western blotting, we confirmed that the engineered bacterial endophyte was successfully constructed, and it harboured insecticidal function after the bioassay in planta. The toxicity of the expressed insecticidal protein was analysed on second instar silkworm. The regression equation showed that the median lethal concentration (LC 50) of the insecticidal protein was 0.77 (0.57-1.04) g/L at 72 h. The insecticidal bacteria genetically modified in this study have laid the foundation for further exploitation of biocontrol bacteria.

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Section: Generation Of Recombinant Plasmidsmentioning

confidence: 99%

Engineering the bacterial endophyte Burkholderia pyrrocinia JK-SH007 for the control of lepidoptera larvae by introducing the cry218 genes of Bacillus thuringiensis

Yang

Zhong

et al. 2017

Biotechnology & Biotechnological Equipment

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A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes

Biedendieck

2016

Advanced Technologies for Protein Complex Production and Characterization

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For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

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Chapter 5 Thermostable Enzymes and Their Industrial Applications

Kumar¹,

Arumugam²,

Permaul³

et al. 2016

Microbial Biotechnology

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Seamless GFP and GFP-Amylase Cloning in Gateway Shuttle Vector, Expression of the Recombinant Proteins inE. ColiandBacillus Megateriumand Assessment of the GFP-Amylase Thermostability

Cited by 3 publications

References 28 publications

Engineering the bacterial endophyte Burkholderia pyrrocinia JK-SH007 for the control of lepidoptera larvae by introducing the cry218 genes of Bacillus thuringiensis

Engineering the bacterial endophyte Burkholderia pyrrocinia JK-SH007 for the control of lepidoptera larvae by introducing the cry218 genes of Bacillus thuringiensis

A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes

Chapter 5 Thermostable Enzymes and Their Industrial Applications

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