Colony PCR

Bergkessel, Megan; Guthrie, Christine

doi:10.1016/b978-0-12-418687-3.00025-2

Cited by 84 publications

(54 citation statements)

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“…Selection of positive transformant was performed on LB / Ampicilin / IPTG / X-Gal selection media. Confirmation of successful transformation was done by colony PCR method (Bergkessel and Guthrie, 2013). The pGEM-T-Easy vector carrying bglp15.2INU and bglp15.2 were isolated from positive transformant using ATP® Plasmid Mini Kit from ATP Biotech, Inc..…”

Section: Methodsmentioning

confidence: 99%

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Fitri¹,

Restiawaty

Moeis³

2017

Jurnal Biodjati

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One of the important enzymes in cellulase complex is β-glucosidase. In this research, adding signal peptide of inulinase gene from Kluyveromyces marxianus, cloning, and expressing of bglp15.2 gene in S. cerevisiae BY4741 had been done. Gene of bglp15.2 encoding β-glucosidase has 90% identity to nucleotide sequence of Shewanella frigidimarina NCIMB 400 bacteria. Adding nucleotide sequence of signal peptide was aimed to secrete β-glucosidase and had been done with PCR (Polymerase Chain Reaction) method. The addition of nucleotide sequence of signal peptide in bglp15.2 gene had been done succesfully that indicated from nucleotide sequencing result and the increment of amplicon band size in electroferogram of the last addition PCR step. The bglp15.2 and bglp15.2INU gene (the bglp15.2 gene that has signal peptide nucleotide sequence) were cloned in Escherichia coli DH5α using pGEM-T-Easy vector and pBEVY-GL shuttle vector. The pBEVY-GL shuttle vector was used for transforming S. cerevisiae BY4741 with bglp15.2 and bglp15.2INU. The recombinant S. cerevisiae BY4741 carrying bglp15.2INU gene and growing in 48 hours had extracellularly β-glucosidase enzyme activity of 0,0178 U/ml and the intracellularly activity was 0,0181 U/ml. The β-glucosidase enzyme without signal peptide was not secreted. With K. marxianus inulinase signal peptide, about 50% Bglp15.2INU protein could be secreted. The protein molecular weight of secreted Bglp15.2INU was 44 kDa in SDS-PAGE result.

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Section: Methodsmentioning

confidence: 99%

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Fitri¹,

Restiawaty

Moeis³

2017

Jurnal Biodjati

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“…DNA was extracted and amplified according to Bergkessel and Guthrie (2013). A region of about 1,400 bp from the 16S rRNA gene was amplified using the primers F8 (5 ′ -AGAGTTTGATCCTGGCTCAG-3 ′ ) and R1492 (5 ′ -GGTTACCTTGTTACGACTT-3 ′ ).…”

Section: Isolation and Characterization Of Bacterial Strainsmentioning

confidence: 99%

Effects of the Ionizing Radiation Disinfection Treatment on Historical Leather

et al. 2020

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Microorganisms often cause significant damage on historical objects. The archive or library materials as well as textile or leather artifacts suffer serious attacks that need appropriate care treatments. Several biocide processes have been implemented but often their application does not preserve the material of the good. The objective of this work is the disinfection through ionizing radiation of leather wallpaper from the museum building Palazzo Chigi in Ariccia (Rome, Italy). The controlled sterilization treatments were carried out using X-ray beams to eliminate the microorganisms present on the leather and maintaining unchanged the properties of the constituent material. Some fragments of decorated leather wallpaper, dating back to the 1700s, were irradiated with X-rays up to 5,000 Gy. The amount of microorganisms was evaluated by microbiological analysis before and after X-ray irradiation treatments to identify the dose that inhibits the bacterial load. It will be shown how the results obtained by the application of different chemical-physical techniques (Scanning Electron Microscopy, Fourier Transform Infrared spectroscopy and Light Transmission Analysis) have helped in the evaluation of the impact of the X-rays on leather chemical and physical integrity.

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“…Different bacteria were isolated from the hypogeum wall in Basilica di San Nicola in Carcere Church, in Rome. Molecular characterization of the isolated bacteria was performed by DNA extraction according to Bergkessel and Guthrie (2013) and the amplification of a region of approximately 1,400 bp from the 16S rRNA gene using the primers F8 (5 -AGAGTTTGATCCTGGCTCAG-3 ) and R1492 (5 -GGTTACCTTGTTACGACTT-3 ). The PCR reaction was performed utilizing Taq DNA polymerase from Accuzyme DNA Polymerase (Bioline).…”

Section: Isolation and Characterization Of Bacterial Strainsmentioning

confidence: 99%

Nanopore Sequencing and Bioinformatics for Rapidly Identifying Cultural Heritage Spoilage Microorganisms

et al. 2020

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Microbiological methodologies allow understanding the causes that lead to the development of a certain microbial community colonizing an artistic surface, to characterize its composition and describe its role in the deterioration of the constituent materials. Metagenomics allows identifying microbial communities directly in their natural environments, bypassing the need for isolation and cultivation of individual species, thus providing a more comprehensive picture of the biodiversity present on a surface compared with standard cultivation methods. Furthermore, molecular analyses require small amounts of material, favoring the preservation of the artistic surface during sampling. Here, we verified the suitability of a protocol consisting in DNA extraction with micro-invasive sampling, using adhesive tape, PCR amplification with universal primers [bacteria (16S), fungi (ITS), and Viridiplantae (18S)], and amplicon sequencing by Oxford Nanopore Technologies (ONT) in the hypogeum of Basilica di San Nicola in Carcere Church (Rome, Italy). Sequence data were analyzed with a bioinformatic pipeline customized for pinpointing cultural heritage spoiling organisms, named "AmpLIcon SequencIng Analysis" (ALISIA). These data were integrated with traditional microbiology techniques that allowed the isolation of cultivable bacteria; three species were also characterized through their capability of biofilm formation and antibiotic resistance. Further, Fourier-transform infrared spectroscopy (FTIR) spectroscopy was performed to characterize the main products present on the masonry surface providing indications on the type of decay present. This novel biological workflow represents a powerful opportunity to investigate the microbial colonization of artistic surfaces aimed at implementing preservation strategies of cultural heritage from bio-spoilage.

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Colony PCR

Cited by 84 publications

References 0 publications

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Extracellular β-Glucosidase Production from bglp15.2 Gene Carrying Inulinase Signal Peptide in Saccharomyces cerevisiae BY4741

Effects of the Ionizing Radiation Disinfection Treatment on Historical Leather

Nanopore Sequencing and Bioinformatics for Rapidly Identifying Cultural Heritage Spoilage Microorganisms

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