Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors

1985 ◽  
Vol 5 (2) ◽  
pp. 101-111 ◽  
Author(s):  
Tuyen Vo-Quang ◽  
Yves Malpiece ◽  
Dominique Buffard ◽  
P. Alexandre Kaminski ◽  
Dominique Vidal ◽  
...  
Keyword(s):  
Large Scale ◽  
Recombinant Dna ◽  
Gel Filtration ◽  
Low Pressure ◽  
Expression Vectors ◽  
Recombinant Dna Technology ◽  
Hybrid Proteins ◽  
Protein Contamination ◽  
As Adsorption ◽  
Foreign Genes

This paper describes a new method of lasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultra-centrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery oI plasmids: (I) in preparative quantities (from 300 gg to 4 mg), (II) exempt from RNA, DNA and protein contamination, and (III) suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purilication, the plasmids were used to construct thermoampliIiable vectors, carrying the tacUV5 promoter and the 5′ end of the β -gallactosidase gone with a single EcoRl site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.

Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

2008 ◽  
Vol 59 (11) ◽  
Author(s):  
Iulia Lupan ◽  
Sergiu Chira ◽  
Maria Chiriac ◽  
Nicolae Palibroda ◽  
Octavian Popescu
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Enzymatic Synthesis ◽  
Large Scale ◽  
Recombinant Dna ◽  
Industrial Enzymes ◽  
Recombinant Dna Technology ◽  
Bacterial Fermentation ◽  
Natural Protein ◽  
Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

scholarly journals Development Of A Gradient System For The Establishment Of Cellulolytic Microbial Communities

2021 ◽  
Author(s):  
Ashley Sousa
Keyword(s):  
Large Scale ◽  
Recombinant Dna ◽  
Consolidated Bioprocessing ◽  
Environmental Gradients ◽  
Industrial Applications ◽  
Single Step ◽  
Gradient System ◽  
Recombinant Dna Technology ◽  
Selective Enrichment ◽  
Feasible Alternative

Cellulosic ethanol has shown promise as a feasible alternative fuel, especially if the hydrolysis of lignocellulosic biomass is done through a single step process known as consolidated bioprocessing (CBP). A major challenge for CBP, especially for large-scale industrial applications is the inhibition of celluloytic microorganisms by ethanol. While recombinant DNA technology and microbial acclimatization by exposure have resulted in some increase in ethanol tolerance, the search remains for robust bacteria that can proliferate in industrially-relevant conditions. This study applied an anaerobic gradient system to provide a continous spatial pathway for the selection of cellulolytic consortia with increased tolerance to ethanol. DGGE analysis showed that increasing concentrations of ethanol impacts the community profile. Biofilm formation of cellulose degrading communities has been found to be influenced by species diversity. Environmental gradients have shown promise for selective enrichment of cellulolytic consortia at desired conditions required for industrial application.

scholarly journals Microbial insecticides in forestry

1991 ◽  
Vol 67 (5) ◽  
pp. 473-480 ◽  
Author(s):  
J. C. Cunningham ◽  
K. van Frankenhuyzen
Keyword(s):  
Recombinant Dna ◽  
Insect Pests ◽  
Genetic Manipulation ◽  
Microbial Control ◽  
Control Agent ◽  
Recombinant Dna Technology ◽  
Pine Sawfly ◽  
Foreign Genes ◽  
Bacterium Bacillus ◽  
European Pine Sawfly

Research has been conducted in Canada on bacteria, viruses, protozoa, fungi and nematodes for control of forest insect pests. Environmental concerns regarding the use of synthetic chemical pesticides have resulted in increased use of the only microbial control agent that is commercially available, the bacterium Bacillus thuringiensis (B.t.). There are currently 18 B.t. products registered for forestry use in Canada. The greatest use of B.t. has been for control of spruce budworm, Choristoneura Jumiferana, although it has been extensively used on several other species of defoliating lepidopterous pests.The use of other microbial control agents is insignificant compared to B.t. Three viral insecticides containing baculoviruses are registered in Canada, two for control of Douglas-fir tussock moth and one for control of redheaded pine sawfly. Registration petitions have been submitted for viral insecticides to control European pine sawfly and gypsy moth.The advent of recombinant DNA technology has opened the door to limitless possibilities for the genetic manipulation of microbial insecticides. Genetic engineering of B.t. toxin genes into other microorganisms and into plants has been accomplished. Foreign genes have been expressed in baculoviruses; most of these products have pharmaceutical applications unrelated to insect control, but this technology can be used to engineer viral insecticides for enhanced activity.

scholarly journals Development Of A Gradient System For The Establishment Of Cellulolytic Microbial Communities

2021 ◽  
Author(s):  
Ashley Sousa
Keyword(s):  
Large Scale ◽  
Recombinant Dna ◽  
Consolidated Bioprocessing ◽  
Environmental Gradients ◽  
Industrial Applications ◽  
Single Step ◽  
Gradient System ◽  
Recombinant Dna Technology ◽  
Selective Enrichment ◽  
Feasible Alternative

Cellulosic ethanol has shown promise as a feasible alternative fuel, especially if the hydrolysis of lignocellulosic biomass is done through a single step process known as consolidated bioprocessing (CBP). A major challenge for CBP, especially for large-scale industrial applications is the inhibition of celluloytic microorganisms by ethanol. While recombinant DNA technology and microbial acclimatization by exposure have resulted in some increase in ethanol tolerance, the search remains for robust bacteria that can proliferate in industrially-relevant conditions. This study applied an anaerobic gradient system to provide a continous spatial pathway for the selection of cellulolytic consortia with increased tolerance to ethanol. DGGE analysis showed that increasing concentrations of ethanol impacts the community profile. Biofilm formation of cellulose degrading communities has been found to be influenced by species diversity. Environmental gradients have shown promise for selective enrichment of cellulolytic consortia at desired conditions required for industrial application.

Based on DNA OTP Key Generation and Management Research

2013 ◽  
Vol 427-429 ◽  
pp. 2470-2472
Author(s):  
Yun Peng Zhang ◽  
Feng Ying Tian ◽  
Man Hui Sun ◽  
Ding Yu ◽  
Fei Xiang Fan ◽  
...  
Keyword(s):  
Data Storage ◽  
Technology Use ◽  
Recombinant Plasmid ◽  
Large Scale ◽  
Recombinant Dna ◽  
Restriction Enzymes ◽  
Key Generation ◽  
Recombinant Dna Technology ◽  
New Approach ◽  
Large Scale Data

With the development of molecular-bio technology, the feature of DNA molecules for ultra-large-scale data storage has created a new approach for data storage. This paper gives a way of strengthening key transport security. Through recombinant DNA technology, use only sender-receiver know restriction enzymes to combine the key DNA and the T vector, to form a recombinant plasmid, making the key DNA bio-hide, and then place the recombinant plasmid in implanted bacteria .

Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

1987 ◽  
Vol 45 ◽  
pp. 924-925
Author(s):  
F. A. Durum ◽  
R. G. Goldman ◽  
T. J. Bolling ◽  
M. F. Miller
Keyword(s):  
Inclusion Bodies ◽  
Large Scale ◽  
Recombinant Dna ◽  
Test System ◽  
Cell Protein ◽  
Gram Negative Bacteria ◽  
Cytoplasmic Inclusions ◽  
Isolation And Purification ◽  
E Coli ◽  
Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

scholarly journals Protein-Engineered Polymers Functionalized with Antimicrobial Peptides for the Development of Active Surfaces

2021 ◽  
Vol 11 (12) ◽  
pp. 5352
Author(s):  
Ana Margarida Pereira ◽  
Diana Gomes ◽  
André da Costa ◽  
Simoni Campos Dias ◽  
Margarida Casal ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Recombinant Dna ◽  
Biological Properties ◽  
Resistant Bacteria ◽  
Recombinant Dna Technology ◽  
Free Standing ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Antimicrobial Materials

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

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