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.

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

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.

Sensitivity of Escherichia coli to Viral Nucleic Acid. XVI. Temperature Conditions for Ca2+-Dependent DNA Uptake in Escherichia coli

1982 ◽  
Vol 37 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Akira Taketo
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Recipient Cell ◽  
Heat Pulse ◽  
Cell Complex ◽  
Dna Uptake ◽  
Viral Nucleic Acid ◽  
E Coli ◽  
Process Formation ◽  
Uptake Process

Properties of Ca2+- or Ba2+-dependent transfection and transformation in Escherichia coli were examined, using ΦX174 replicative-form (RF) DNA and plasmid DNA. For the transfection and transformation, a heat pulse step was dispensable and the yield of transfectants was, in most E. coli strains, rather reduced by the heat treatment. The heat pulse step was also detrimental for the transformation of certain strains such as lipopolysaccharide mutants. The first stage of the DNA uptake process (formation of DNA · recipient cell complex) was dependent on low temperature and Ca2+ ion. A substantial amount of the complexed RF-DNA was released from the bacteria, by washing with a chilled Tris buffer. Although a RF-DNA · cell complex was formed even at 37 °C or in chilled 0.05 ᴍ MgCl2, the complex did not yield transfectants.

Transformation of Acidiphilium by electroporation and conjugation

1992 ◽  
Vol 38 (5) ◽  
pp. 387-393 ◽  
Author(s):  
Anne W. Glenn ◽  
Frank F. Roberto ◽  
Thomas E. Ward
Keyword(s):  
Host Range ◽  
Transformation Efficiency ◽  
Agar Medium ◽  
Recipient Cell ◽  
Coli Strain ◽  
Broad Host Range ◽  
E Coli ◽  
Acidophilic Bacteria ◽  
Nutrient Agar Medium ◽  
Broad Host Range Plasmids

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigaed. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 °C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 °C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10–15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 μg/mL. Transformation efficiencies in these experiments ranged up to 104 transformants/μg DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10−5–10−9 per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain. Key words: acidophilic bacteria, endogenous plasmids, broad-host-range plasmids.

scholarly journals Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

scholarly journals Agrobacterium tumefaciens Deploys a Versatile Antibacterial Strategy To Increase Its Competitiveness

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Manda Yu ◽  
Yi-Chieh Wang ◽  
Ching-Jou Huang ◽  
Lay-Sun Ma ◽  
Erh-Min Lai
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Environmental Conditions ◽  
Recipient Cell ◽  
Biological Significance ◽  
Carbon Starvation ◽  
Growth Suppression ◽  
Diaminopimelic Acid ◽  
Type Vi Secretion System ◽  
Content Type ◽  
E Coli

ABSTRACT The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the effectors in the system share the same purpose of target intoxication, but the rationale for maintaining various types of effectors in a species is not well studied. In this study, we showed that a peptidoglycan amidase effector in Agrobacterium tumefaciens, Tae, cleaves d-Ala-meso-diaminopimelic acid (mDAP) and d-Glu bonds in peptidoglycan and is able to suppress the growth of Escherichia coli recipient cells. The growth suppression was effective only under the condition in which E. coli cells are actively growing. In contrast, the Tde DNase effectors in the strain possessed a dominant killing effect under carbon starvation. Microscopic analysis showed that Tde triggers cell elongation and DNA degradation, while Tae causes cell enlargement without DNA damage in E. coli recipient cells. In a rich medium, A. tumefaciens harboring only functional Tae was able to maintain competitiveness among E. coli and its own sibling cells. Growth suppression and the competitive advantage of A. tumefaciens were abrogated when recipient cells produced the Tae-specific immunity protein Tai. Given that Tae is highly conserved among A. tumefaciens strains, the combination of Tae and Tde effectors could allow A. tumefaciens to better compete with various competitors by increasing its survival during changing environmental conditions. IMPORTANCE The T6SS encodes multiple effectors with diverse functions, but little is known about the biological significance of harboring such a repertoire of effectors. We reported that the T6SS antibacterial activity of the plant pathogen Agrobacterium tumefaciens can be enhanced under carbon starvation or when recipient cell wall peptidoglycan is disturbed. This led to a newly discovered role for the T6SS peptidoglycan amidase Tae effector in providing a growth advantage dependent on the growth status of the target cell. This is in contrast to the Tde DNase effectors that are dominant during carbon starvation. Our study suggests that combining Tae and other effectors could allow A. tumefaciens to increase its competitiveness among changing environmental conditions.

scholarly journals Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jun Ren ◽  
Hyang-Mi Lee ◽  
Thi Duc Thai ◽  
Dokyun Na
Keyword(s):  
Genetic Engineering ◽  
Plasmid Dna ◽  
Metabolic Flux ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Value Added ◽  
Cytosine Methyltransferase ◽  
Genome Bisulfite Sequencing ◽  
First Time ◽  
Value Added Products

Abstract Background Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism. Results We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA. Conclusions Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

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