Preparation of Competent Cells for High-Efficiency Plasmid Transformation of Escherichia coli

Nakata, Yasuhiko; Tang, Xiaoou; Yokoyama, Kazunari K.

doi:10.1385/0-89603-383-x:129

Cited by 12 publications

(10 citation statements)

References 2 publications

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“…Ligation step was followed by the action of T4-DNA ligase enzyme (New England Biolabs (NEB), USA) with 3:1 ratio of insert and plasmid respectively. The transformation of the cloned plasmid into E. coli (DH5α) competent cells were accomplished by heat shock at 42°C for 30 s followed by immediately transferring to ice for 2 min as mentioned in Nakata et al (1997) protocol followed by streaking the transformed cells over Luria agar (LB) (Lab M®, UK) containing 100 μg/ ml of ampicillin and incubated overnight at 37°C. The positive colonies harbored the recombinant plasmid and the negative colonies harbored the empty plasmid.…”

Section: Cloningmentioning

confidence: 99%

Expression, purification and testing of zinc metalloproteinase aureolysin as potential vaccine candidate against Staphylococcus aureus

Solyman¹,

Reda²,

Hanora³

2021

Afr. J. Biotechnol.

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Staphylococcus aureus (S. aureus) is a versatile bacterium which exhibits multiple antibiotic resistances. To ameliorate the undesirable diseases causing potential, there is a need to design a protective vaccine capable of stimulating immune response against this pathogen. In a similar study in our laboratory, reverse vaccinology approach was used to nominate potential vaccine candidate genes against S. aureus. Zinc Metalloproteinase Aureolysin (aur) gene was one of the nominated genes based on that previously published in-silico study. The objective of this study is the cloning, expression, purification of aur gene and testing the aur protein reactivity with serum antibodies collected from groups of human patients with confirmed Staphylococcal disease. Cloning was done in pH6HTN His6HaloTag® vector and it was expressed in E. coli BL21 (DE3) using these conditions; 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37°C. Purification was carried out by Immobilized Metal Affinity Chromatography (IMAC). The his-tag aur protein was detected at ~86 KDa as a single band after western blot assay and was successfully reacted with antibodies obtained from humans infected with S. aureus. The results encourage further testing of aur protein as a potential vaccine candidate for S. aureus.

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Section: Cloningmentioning

confidence: 99%

Expression, purification and testing of zinc metalloproteinase aureolysin as potential vaccine candidate against Staphylococcus aureus

Solyman¹,

Reda²,

Hanora³

2021

Afr. J. Biotechnol.

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“…The site directed mutagenesis PCR protocol included an initial denaturation (30 s, 98°C), followed by 25 cycles (each 10 s at 98°C, 30 s at a primer specific annealing temperature (Table S4), and 95 s at 72°C) and a final extension (2 min, 72°C). pPICZαA-HvLD wild type and variants were transformed into E. coli DH5α [49] using heat shock [50] and selected with 25 µg mL -1 zeocin (Invitrogen). Mutant plasmids were verified by sequencing (GATC Biotech, Konstanz, GER), linearized by PmeI (20 units; New England Biolabs), purified (GeneJet PCR Purification Kit; Fermentas, Waltham, USA), and precipitated (Pellet Paint® Co-Precipitant; Merck Millipore, Billerica, USA) yielding about 3 µg linearized plasmid.…”

Section: Mutagenesis and Pichia Pastoris Transformationmentioning

confidence: 99%

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

Andersen

Svensson

Møller

2020

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…More important is that the transformation efficiency is much lower than that of electroporation, which renders it unsuitable for several molecular cloning experiments such as the construction of high-complexity cDNA libraries with a minimum expenditure of mRNA [11]. A great number of studies have make efforts toward establishing a quick and efficient method for the preparation of such competent E. coli cells with an extremely high transforming frequency, which meets the requirements of modern molecular cloning experiments [3,[12][13][14][15][16]. There are two avenues to improving the preparation protocol, the first is simplifying the steps and shorting preparation time and the second is increasing the transforming frequency of chemically competent cells [11,[17][18][19][20][21].…”

Section: Introductionmentioning

confidence: 99%

An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments

et al. 2018

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The high-throughput, cost-efficient transformation systems determine the success of gene cloning and functional analysis. Among various factors that affect this transformation systems, the competence ability of target cells is one of the most important factors. We found antimicrobial peptides LFcin-B can increase the permeability of the cell membrane, and their lethal antibacterial properties can be inhibited by moderately high concentrations of Ca and Mn. In this study, we established a convenient and rapid method (CRM) by adding small concentrations of (0.35 mg/L) and moderately high concentrations of MnCl (50 mM) and CaCl (30 mM) in transformation buffer. The transformation efficiency of cells (DH5α, JM109 and TOP10) prepared by CRM were comparable with electroporation for plasmid transformation (3.1 ± 0.3 × 10 cfu/µg). Unlike competent cells prepared using other chemical methods, those obtained using CRM method are extremely competent for receiving larger size DNA fragments (> 5000 bp) into plasmid vectors. The competent cells prepared by CRM method are particularly useful for most high-efficiency transformation experiments under normal laboratory conditions.

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Preparation of Competent Cells for High-Efficiency Plasmid Transformation of Escherichia coli

Cited by 12 publications

References 2 publications

Expression, purification and testing of zinc metalloproteinase aureolysin as potential vaccine candidate against Staphylococcus aureus

Expression, purification and testing of zinc metalloproteinase aureolysin as potential vaccine candidate against Staphylococcus aureus

Roles of the N-terminal domain and remote substrate binding subsites in activity of the debranching barley limit dextrinase

An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments

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