Influence of genetic background on transformation and expression of Green Fluorescent Protein in <i>Actinobacillus actinomycetemcomitans</i>

Teughels, Wim; Sliepen, I.; Keersmaecker, Sigrid C. J. De; Quirynen, Marc; Lippmann, Joan E.; Pauwels, Martine; Fives-Taylor, Paula

doi:10.1111/j.1399-302x.2005.00224.x

Cited by 3 publications

(2 citation statements)

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“…This serotype is strongly associated with periodontal disease (28) and is the most common serotype found in patients with aggressive periodontitis (27). Bacteria were transformed by electroporation (24) with a GFP expression vector (20) to allow differentiation in the presence of other bacterial species and/or epithelial vesicles or other similar structures. Average fluorescence intensity was used as a marker for biomass accumulation because the greater the accumulation, the higher the intensity (4).…”

Section: Discussionmentioning

confidence: 99%

Colonization of hard and soft surfaces by Aggregatibacter actinomycetemcomitans under hydrodynamic conditions

Sliepen

Essche

Pauwels

et al. 2008

Oral Microbiology and Immunology

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Section: Discussionmentioning

confidence: 99%

Colonization of hard and soft surfaces by Aggregatibacter actinomycetemcomitans under hydrodynamic conditions

Sliepen

Essche

Pauwels

et al. 2008

Oral Microbiology and Immunology

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“…Electro-transformation is an effective artificial transformation method that introduces natural or recombinant foreign protein-encoding genes into microorganisms for overexpression or homologous recombination for gene knockout or knock-in [ 30 , 31 ]. It is widely used in the directional transformation of fungi and bacterial strains [ 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

Stable transformation of fluorescent proteins into Nosema bombycis by electroporation

et al. 2022

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Background Microsporidia are a group of intracellular parasitic eukaryotes, serious pathogens that cause widespread infection in humans, vertebrates, and invertebrates. Because microsporidia have a thick spore wall structure, the in vitro transformation, cell culture, and genetic operation technology of microsporidia are far behind that of other parasites. Methods In this study, according to an analysis of the life-cycle of microsporidia, Nosema bombycis, and different electro-transformation conditions, the transduction efficiency of introducing foreign genes into N. bombycis was systematically determined. Results We analyzed the direct electro-transformation of foreign genes into germinating N. bombycis using reporters under the regulation of different characteristic promoters. Furthermore, we systematically determined the efficiency of electro-transformation into N. bombycis under different electro-transformation conditions and different developmental stages through an analysis of the whole life-cycle of N. bombycis. These results revealed that foreign genes could be effectively introduced through a perforation voltage of 100 V pulsed for 15 ms during the period of N. bombycis sporeplasm proliferation. Conclusions We present an effective method for electro-transformation of a plasmid encoding a fluorescent protein into N. bombycis, which provides new insight for establishing genetic modifications and potential applications in these intracellular parasites. Graphical Abstract

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Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

2016

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Green fluorescent protein (GFP) is one of the most useful biological markers for monitoring gene expression and the localization of particular cells. We constructed a GFPexpressing Aggregatibacter actinomycetemcomitans transformant by transposon insertion.This anaerobic, oral, gram-negative bacterium is thought to be the major causative agent in localized aggressive periodontitis and possesses the gene coding for leukotoxin (ltx). As this gene is stably expressed, the region extending from the ltx promoter to the initiation codon was amplified using PCR and temporarily cloned into a small Escherichia coli plasmid, pResKm. The DNA fragment containing the GFP gene(gfp)ORF was excised from the commercially available plasmid pAcGFP1-C1, purified by gel electrophoresis, and inserted downstream of the ltx initiation codon in the correct reading frame. This gene fusion was further cloned into the NotI site of the commercially available shuttle plasmid, pUTminiTn5, which contains the gene coding for the transposase, and then introduced into the chromosome of A. actinomycetemcomitans strain HK1651 by electroporation.Transformants were isolated by screening for the appropriate antibiotic resistance. FACS analysis revealed that the final A. actinomycetemcomitans-GFP integrant exhibited slightly more intense fluorescence than wild-type A. actinomycetemcomitans. These data demonstrate that transposon-mediated chromosomal integration is a useful approach for constructing mutant A. actinomycetemcomitans strains.

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Influence of genetic background on transformation and expression of Green Fluorescent Protein in Actinobacillus actinomycetemcomitans

Abstract: The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent.

Cited by 3 publications

References 47 publications

Colonization of hard and soft surfaces by Aggregatibacter actinomycetemcomitans under hydrodynamic conditions

Colonization of hard and soft surfaces by Aggregatibacter actinomycetemcomitans under hydrodynamic conditions

Stable transformation of fluorescent proteins into Nosema bombycis by electroporation

Transformation of <i>Aggregatibacter actinomycetemcomitans</i> with Green Fluorescent Protein

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