Optimization of E. coli culture conditions for efficient DNA uptake by electroporation

Ahmad, Irfan; Rubbab, Tehseen; Deeba, Farah; Naqvi, Syed Muhammad Saqlan

doi:10.3906/biy-1311-60

Cited by 6 publications

(3 citation statements)

References 25 publications

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“…This inverse proportion between CaCl 2 concentration and transformation efficiency may be due to lipid array on cell membrane is destroyed by 75 to 100 mM Ca 2+ , and then, a liquid crystal would be formed. The bacteria would be swollen when incubated in hypotonic calcium chloride solution, at 0 °C, and DNA in the mixture can be formed into hydroxyapatite (anti-DNase) and then stick to the surface of cells [ 27 , 28 ]. The cellular absorption ability of exogenous DNA will be increased after heat shock at 42 °C.…”

Section: Discussionmentioning

confidence: 99%

Molecular docking and nucleotide sequencing of successive expressed recombinant fungal peroxidase gene in E.coli

Khedr

Khalil

Kabary

et al. 2022

Journal of Genetic Engineering and Biotechnology

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Background Fungal peroxidases are oxidoreductases that utilize hydrogen peroxide to catalyze lignin biodegradation. Results PER-K (peroxidase synthesis codon gene) was transformed from Aspergillus niger strain AN512 deposited in the National Center for Biotechnology Information with the accession number OK323140 to Escherichia coli strain (BL21-T7 with YEp356R recombinant plasmid) via calcium chloride heat-shock method. The impact of four parameters (CaCl2 concentrations, centrifugation time, shaking speed, growth intensity) on the efficacy of the transformation process was evaluated. Furthermore, peroxidase production after optimization was assessed both qualitatively and quantitatively, as well as SDS-PAGE analysis. The optimum conditions for a successful transformation process were as follows: CaCl2 concentrations (50 mM), centrifugation time (20 min), shaking speed (200 rpm), and growth optical density (0.45). PCR and gel electrophoresis detect DNA bands with lengths 175, 179, and 211 bps corresponding to UA3, AmpR, and PER-K genes respectively besides partially sequencing the PER-K gene. Pyrogallol/hydrogen peroxide assay confirmed peroxidase production, and the activity of the enzyme was determined to be 3924 U/L. SDS-PAGE analysis also confirms peroxidase production illustrated by the appearance of a single peroxidase protein band after staining with Coomassie blue R-250. Conclusion A successful peroxidase-gene (PER-K) transformation from fungi to bacteria was performed correctly. The enzyme activity was screened, and partial sequencing of PER-K gene was analyzed successively. The protein 3D structure was generated via in silico homology modeling, and determination of binding sites and biological annotations of the constructed protein were carried out via COACH and COFACTOR based on the I-TASSER structure prediction.

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Section: Discussionmentioning

confidence: 99%

Molecular docking and nucleotide sequencing of successive expressed recombinant fungal peroxidase gene in E.coli

Khedr

Khalil

Kabary

et al. 2022

Journal of Genetic Engineering and Biotechnology

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“…The resulting PCR fragment was ligated to BamHl and Pstl sites next to merR in pUMER after predigestions with BamHl and Pstl enzymes. The final 3.96-kb construct was transformed into the E. coli strains DH5α and BL21 (DE3) (Ahmad et al, 2014). 2.2.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Designing a bacterial biosensor for detection of mercury in water solutions

Roointan¹,

Shabab²,

Karimi³

et al. 2015

Turk J Biol

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Due to increasing advances in physical and chemical techniques for the assessment of pollutants in the environment, there is an immediate demand for a bioassay that can report both the presence of an analyte and its biological effects. In accordance with this need, there has been a fast growth in whole-cell biosensor technology. In this study we aimed to design a whole-cell bacterial biosensor to detect mercury in liquid solutions. The Pseudomonas pBS228 merR gene and its related promoter/operator was synthesized by Bioneer. The green fluorescence protein (GFP) gene was used as a reporter. GFP was cloned downstream of the merR gene. The construct, including the merR promoter, gene, and GFP, was cloned in a pUC19 vector and transferred into E. coli BL21 (DE3) bacteria. Transformed bacteria were used as whole-cell biosensors for detecting mercury. Mercury detection was monitored by means of microscopy and fluorometry techniques. Transformed BL21 (DE3) biosensors responded mainly to Hg(II), with the lowest detectable concentration being 10 -8 M during a 3-h exposure induction period. Our results demonstrated that the noninfectious bacterial biosensors developed in the present study could be beneficial and enforceable in detection of mercury in contaminated water samples at concentrations as low as 10 -8 M.

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“…Examples of therapeutic proteins that have utilized CHO for their production include β-interferon, factor VIII, and erythropoietin (Kelley, 2001). Escherichia coli (E.coli) as a microbial host has been also employed for the production of different types of proteins (Kamionka, 2011;Gökbulut and Arslanoğlu, 2013;Ahmad et al, 2014;Wang et al, 2014). S. cerevisiae and P. pastoris are also known to be good microbial hosts for better expression and production of therapeutic proteins.…”

Section: Production and Purification Of Therapeutic Proteinsmentioning

confidence: 99%

Development of therapeutic proteins: advances and challenges

Akash¹,

Rehman²,

Tariq³

et al. 2015

Turk J Biol

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Therapeutic proteins have shown to be effective against a variety of diseases. Pharmaceutical companies are progressively focusing on research using proteins in search of novel effective therapeutics. The significant attention by researchers has resulted in considerable advances in the production and usage of therapeutic proteins in recent years. In the current study we focused on the understanding of therapeutic proteins and their impact on the human health care system. Advancements in the field of biotechnology have increased and facilitated the production of therapeutically significant proteins to combat various fatal diseases. Although proteinbased therapeutics have taken center stage in drug discovery and development and enhanced safety for human beings, certain challenges remain, including safety and immunogenicity issues, protein stability, and degradation issues. We made an attempt to discuss all possible factors that are linked with the basis of therapeutic proteins and possible challenges that are currently being faced by scientists during the development of these therapeutic proteins.

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Optimization of E. coli culture conditions for efficient DNA uptake by electroporation

Cited by 6 publications

References 25 publications

Molecular docking and nucleotide sequencing of successive expressed recombinant fungal peroxidase gene in E.coli

Molecular docking and nucleotide sequencing of successive expressed recombinant fungal peroxidase gene in E.coli

Designing a bacterial biosensor for detection of mercury in water solutions

Development of therapeutic proteins: advances and challenges

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