scholarly journals Development of strategy for competent cell preparation and high efficiency plasmid transformation using different methods

2011 ◽  
Vol 29 (1) ◽  
pp. 17 ◽  
Author(s):  
Anubhuti Sharma ◽  
Arushi Girdhar ◽  
Nidhi Srivastava
Keyword(s):  
High Efficiency ◽  
Transformation Efficiency ◽  
Great Influence ◽  
Competent Cell ◽  
Cell Preparation ◽  
Site Specific ◽  
E Coli ◽  
Cost Efficient ◽  
Normal Laboratory ◽  
Complete Genomic

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible. With the recent availability of complete genomic sequences of many organisms and plants, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. This study compares two different methods of preparation of competent cells using two strains of E. coli DH5α and HB101. From results the most efficient strain was found DH5α for cloning as it supports blue white screening utilizing galactosidase activity. The concentration of calcium chloride is another important factor; various concentrations of CaCl2 were tried. Optimum concentration was found to be 75 mM. However PEG also has great influence on transformation efficiency, use of 40% PEG gave the best transformation efficiency. A convenient and rapid method for the genetic transformation of E. coli with appropriate plasmid is proposed which can be utilised for high efficiency transformation in normal laboratory conditions.

scholarly journals Identification of Site-Specific Recombination Genesint and xis of the Rhizobium Temperate Phage 16-3

1999 ◽  
Vol 181 (14) ◽  
pp. 4185-4192 ◽  
Author(s):  
Szabolcs Semsey ◽  
IstvAn Papp ◽  
Zsuzsanna Buzas ◽  
Andras Patthy ◽  
Laszlo Orosz ◽  
...  
Keyword(s):  
Dna Sequences ◽  
High Efficiency ◽  
Rhizobium Meliloti ◽  
Temperate Phage ◽  
Host Chromosome ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Tyrosine Recombinases ◽  
Recombination System

ABSTRACT Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis(prophage excision). We concluded that Int protein of phage16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

Novel Plasmid Transformation Method Mediated by Chrysotile, Sliding Friction, and Elastic Body Exposure

2007 ◽  
Vol 2 ◽  
pp. 117739010700200 ◽  
Author(s):  
Naoto Yoshida ◽  
Toshiaki Nakajima-Kambe ◽  
Kaori Matsuki ◽  
Toshiya Shigeno
Keyword(s):  
Elastic Body ◽  
Plasmid Dna ◽  
Sliding Friction ◽  
Transformation Efficiency ◽  
Recipient Cell ◽  
Reaction Force ◽  
Transformation Method ◽  
Competent Cell ◽  
E Coli ◽  
Exposure Method

Escherichia coli as a plasmid recipient cell was dispersed in a chrysotile colloidal solution, containing chrysotile adsorbed to plasmid DNA (chrysotile-plasmid cell mixture). Following this, the chrysotile-plasmid cell mixture was dropped onto the surface of an elastic body, such as agarose, and treated physically by sliding a polystyrene streak bar over the elastic body to create friction. Plasmid DNA was easily incorporated into E. coli, and antibiotic resistance was conferred by transformation. The transformation efficiency of E. coli cultured in solid medium was greater than that of E. coli cultured in broth. To obtain greater transformation efficiency, we attempted to determine optimal transformation conditions. The following conditions resulted in the greatest transformation efficiency: the recipient cell concentration within the chrysotile-plasmid cell mixture had an optical density greater than or equal to 2 at 550 nm, the vertical reaction force applied to the streak bar was greater than or equal to 40 g, and the rotation speed of the elastic body was greater than or equal to 34 rpm. Under these conditions, we observed a transformation efficiency of 107 per μg plasmid DNA. The advantage of achieving bacterial transformation using the elastic body exposure method is that competent cell preparation of the recipient cell is not required. In addition to E. coli, other Gram negative bacteria are able to acquire plasmid DNA using the elastic body exposure method.

scholarly journals Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

2015 ◽  
Vol 81 (24) ◽  
pp. 8500-8506 ◽  
Author(s):  
Bo Wang ◽  
Jianping Yu ◽  
Weiwen Zhang ◽  
Deirdre R. Meldrum
Keyword(s):  
High Efficiency ◽  
Transformation Efficiency ◽  
Content Type ◽  
E Coli ◽  
Integrative Transformation ◽  
High Transformation ◽  
Host Genetic ◽  
Foreign Dna ◽  
Encoding Genes ◽  
Pcc 6803

ABSTRACTRestriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium,Synechocystissp. strain PCC 6803 (Synechocystis6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e.,sll0729(geneM) andslr0214(geneC), were cloned from the chromosome ofSynechocystis6803 and expressed inEscherichia coliharboring an integration plasmid. After premethylation treatment inE. coli, the integration plasmid was extracted and used for transformation ofSynechocystis6803. The results showed that although expression of methyltransferase M had little impact on the transformation ofSynechocystis6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation ofSynechocystis6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency inSynechocystis6803. Since premethylating foreign DNA prior to transformingSynechocystisavoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation ofSynechocystis6803.

scholarly journals Free fatty acids promote transformation efficiency of yeast

2019 ◽  
Vol 19 (7) ◽  
Author(s):  
Xingpeng Duan ◽  
Xiaojing Ma ◽  
Shengying Li ◽  
Yongjin J Zhou
Keyword(s):  
Transformation Efficiency ◽  
Culture Media ◽  
Cell Factory ◽  
Competent Cell ◽  
Cell Preparation ◽  
Yeast Transformation ◽  
High Transformation Efficiency ◽  
Metabolic Analysis ◽  
High Transformation ◽  
Large Plasmids

ABSTRACT High transformation efficiency is essential in genetic engineering for functional metabolic analysis and cell factory construction, in particular in construction of long biosynthetic pathways with multiple genes. Here, we found that free fatty acid (FFA)-overproducing strain showed higher transformation efficiency in Saccharomyces cerevisiae. We then verified that external supplementation of FFAs, to the culture media for competent cell preparation, improved yeast transformation efficiency significantly. Among all tested FFAs, 0.5 g/L C16:0 FFA worked best on promoting transformation of S. cerevisiae and Komagataella phaffii (previously named as Pichia pastoris). Furthermore, C16:0 FFA improved the assembly efficiency of multiple DNA fragments into large plasmids and genome by 100%, which will facilitate the construction and optimization of multigene-containing long pathways.

scholarly journals Tannic acid-functionalized HEPA filter materials for influenza virus capture

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Subin Kim ◽  
Jinhyo Chung ◽  
Sang Hyun Lee ◽  
Jeong Hyeon Yoon ◽  
Dae-Hyuk Kweon ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Tannic Acid ◽  
High Efficiency ◽  
Influenza Viruses ◽  
Capture Efficiency ◽  
Protective Equipment ◽  
Filter Materials ◽  
Washing Process ◽  
Pathogenic Viruses ◽  
Cost Efficient

AbstractInfluenza, one of the most contagious and infectious diseases, is predominantly transmitted through aerosols, leading to the development of filter-based protective equipment. Though the currently available filters are effective at removing submicron-sized particulates, filter materials with enhanced virus-capture efficiency are still in demand. Coating or chemically modifying filters with molecules capable of binding influenza viruses has received attention as a promising approach for the production of virus-capturing filters. For this purpose, tannic acid (TA), a plant-derived polyphenol, is a promising molecule for filter functionalization because of its antiviral activities and ability to serve as a cost-efficient adhesive for various materials. This study demonstrates the facile preparation of TA-functionalized high-efficiency particulate air (HEPA) filter materials and their efficiency in influenza virus capture. Polypropylene HEPA filter fabrics were coated with TA via a dipping/washing process. The TA-functionalized HEPA filter (TA-HF) exhibits a high in-solution virus capture efficiency of up to 2,723 pfu/mm2 within 10 min, which is almost two orders of magnitude higher than that of non-functionalized filters. This result suggests that the TA-HF is a potent anti-influenza filter that can be used in protective equipment to prevent the spread of pathogenic viruses.

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽  
1984 ◽  
Vol 23 (9) ◽  
pp. 2073-2078 ◽  
Author(s):  
Anup K. Hazra ◽  
Sevilla Detera-Wadleigh ◽  
Samuel H. Wilson
Keyword(s):  
Dna Polymerase ◽  
Large Fragment ◽  
Dna Polymerase I ◽  
Site Specific ◽  
E Coli ◽  
Specific Modification ◽  
Polymerase I

Advances in coupled axial turbine and nonaxisymmetric exhaust volute aerodynamics for turbomachinery

2020 ◽  
pp. 095441002096645
Author(s):  
Jie Gao ◽  
Chunde Tao ◽  
Dongchen Huo ◽  
Guojie Wang
Keyword(s):  
Performance Improvement ◽  
High Efficiency ◽  
Three Dimensional ◽  
Great Influence ◽  
Aerodynamic Characteristics ◽  
Turbine Engines ◽  
Gas Turbine Engines ◽  
Axial Turbine ◽  
Flow Interactions ◽  
And Performance

Marine, industrial, turboprop and turboshaft gas turbine engines use nonaxisymmetric exhaust volutes for flow diffusion and pressure recovery. These processes result in a three-dimensional complex turbulent flow in the exhaust volute. The flows in the axial turbine and nonaxisymmetric exhaust volute are closely coupled and inherently unsteady, and they have a great influence on the turbine and exhaust aerodynamic characteristics. Therefore, it is very necessary to carry out research on coupled axial turbine and nonaxisymmetric exhaust volute aerodynamics, so as to provide reference for the high-efficiency turbine-volute designs. This paper summarizes and analyzes the recent advances in the field of coupled axial turbine and nonaxisymmetric exhaust volute aerodynamics for turbomachinery. This review covers the following topics that are important for turbine and volute coupled designs: (1) flow and loss characteristics of nonaxisymmetric exhaust volutes, (2) flow interactions between axial turbine and nonaxisymmetric exhaust volute, (3) improvement of turbine and volute performance within spatial limitations and (4) research methods of coupled turbine and exhaust volute aerodynamics. The emphasis is placed on the turbine-volute interactions and performance improvement. We also present our own insights regarding the current research trends and the prospects for future developments.

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