Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

2013 ◽  
Vol 246 (11) ◽  
pp. 861-867 ◽  
Author(s):  
Saša Haberl ◽  
Marko Jarc ◽  
Aleš Štrancar ◽  
Matjaž Peterka ◽  
Duša Hodžić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Alkaline Lysis ◽  
Electric Pulses

Plasmid Fingerprinting Profiles of Chicken-Derived Pathogenic Strains of Escherichia coli

2010 ◽  
Vol 178 ◽  
pp. 355-360
Author(s):  
Chun Guang Wang ◽  
Tie Zhang ◽  
Xing Hua Zhao
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Gel Electrophoresis ◽  
Molecular Level ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Epidemiological Investigation ◽  
Alkaline Lysis ◽  
Plasmid Profiles ◽  
Different Sources

Plasmid DNA was extracted from all 38 strains of chicken Escherichia coli by alkaline lysis method. The agarose gel electrophoresis showed 23 plasmid patterns. The strains from the same source had the same, similar, or quite different plasmid profiles, while those from different sources generally did not have the same plasmid profiles. Therefore, the plasmid profiles can provide basic materials for epidemiological investigation on chicken Escherichia coli at molecular level.

scholarly journals Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

2003 ◽  
Vol 185 (3) ◽  
pp. 1097-1100 ◽  
Author(s):  
Yazmid Reyes-Domínguez ◽  
Gabriel Contreras-Ferrat ◽  
Jesús Ramírez-Santos ◽  
Jorge Membrillo-Hernández ◽  
M. Carmen Gómez-Eichelmann
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Plasmid Dna ◽  
Bimodal Distribution ◽  
Dna Supercoiling ◽  
Wild Type

ABSTRACT Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes

1990 ◽  
Vol 10 (7) ◽  
pp. 3505-3511
Author(s):  
J B Hays ◽  
E J Ackerman ◽  
Q S Pang
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Plasmid Dna ◽  
Excision Repair ◽  
Xenopus Laevis Oocytes ◽  
Marked Contrast ◽  
Repair Synthesis ◽  
Error Frequency ◽  
Repair Activity ◽  
Good Agreement

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

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.

Low intensity infrared laser effects on Escherichia coli cultures and plasmid DNA

Laser Physics ◽  
2012 ◽  
Vol 22 (10) ◽  
pp. 1635-1641 ◽  
Author(s):  
A. S. Fonseca ◽  
A. F. Teixeira ◽  
G. A. Presta ◽  
M. Geller ◽  
S. S. Valença ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Infrared Laser ◽  
Low Intensity

scholarly journals Horizontal Transfer of Antimicrobial Resistance by Extended-Spectrum β Lactamase-Producing Enterobacteriaceae

2011 ◽  
Vol 3 (01) ◽  
pp. 037-042 ◽  
Author(s):  
Varsha K Vaidya
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Environmental Conditions ◽  
Horizontal Transfer ◽  
Hospital Setting ◽  
Alkaline Lysis ◽  
Antibiotic Resistant ◽  
Extended Spectrum ◽  
Synergy Test

ABSTRACT Background: The purpose of this work was to study the acquisition of new antibiotic-resistant genes carried by extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae via horizontal transfer to understand their rampant spread in the hospitals and in the community. Materials and Methods: A retrospective analysis of 120 ESBL screen-positive isolates of Escherichia coli and Klebsiella pneumoniae, which were subjected to antimicrobial susceptibility testing, was carried out. The Double Disc Synergy Test (DDST) and Inhibitor-Potentiation Disc Diffusion Test (IPDD) were employed for confirmation of ESBL activity. The transferability of the associated antibiotic resistance for amoxicillin, amikacin, gentamicin, cefotaxime and ceftriaxone was elucidated by intra- and intergenus conjugation in Escherichia coli under laboratory as well as under simulated environmental conditions. Transformation experiments using plasmids isolated by alkaline lysis method were performed to study the transferability of resistance genes in Klebsiella pneumoniae isolates. Results : ESBL production was indicated in 20% each of the Escherichia coli and Klebsiella pneumoniae isolates. All the ESBL isolates showed co- resistance to various other groups of antibiotics, including 3GC antibiotics, though all the isolates were sensitive to both the carbapenems tested. Conjugation-mediated transfer of resistance under laboratory as well as environmental conditions at a frequency of 3-4 x 10-5 , and transformation-mediated dissemination of cefotaxime and gentamicin resistance shed light on the propensity of ESBL producers for horizontal transfer. Conclusions: The transfer of resistant markers indicated availability of a large pool of resistance genes in the hospital setting as well as in the environment, facilitating long-term persistence of organisms.

scholarly journals Inhibition of transcription starting from bacteriophage lambda pR promoter during the stringent response in Escherichia coli: implications for lambda DNA replication.

1995 ◽  
Vol 42 (2) ◽  
pp. 233-239 ◽  
Author(s):  
A Szalewska-Pałasz ◽  
G Wegrzyn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Plasmid Dna ◽  
Bacteriophage Lambda ◽  
Stringent Response ◽  
Lacz Gene ◽  
Relative Level ◽  
Lambda Dna

Replication of lambda plasmid DNA is halted in amino acid-starved wild type (stringent) strains whereas it proceeds in relA (relaxed) mutants. The only transcription which could be important in lambda plasmid DNA replication in amino acid-starved Escherichia coli cells is that starting from the pR promoter. Using a fusion which consists of the lacZ gene under the control of bacteriophage lambda pR promoter we found that transcription starting from this promoter was inhibited during the stringent, but not the relaxed, response in E. coli. We confirmed our conclusion by estimating the relative level of the pR transcript by RNA-DNA hybridization. We propose that decreased transcription from the pR promoter which serves as transcriptional activation of ori lambda is responsible for inhibition of lambda plasmid replication during the stringent response. The results presented in this paper, combined with our recent findings (published elsewhere), indicate that the transcriptional activation of ori lambda may be a main regulatory process controlling lambda DNA replication not only during the relaxed response but also in normal growth conditions.

scholarly journals Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Kuan Yao ◽  
Narjol González-Escalona ◽  
Maria Hoffmann
Keyword(s):  
Antibiotic Resistance ◽  
Single Molecule ◽  
Plasmid Dna ◽  
Copy Number ◽  
Sequence Data ◽  
Multiple Displacement Amplification ◽  
Fast Method ◽  
Alkaline Lysis ◽  
Low Copy Number ◽  
Long Read

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

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