A rapid and simple method for plasmid copy number comparison

Asaka, Orie; Ano, Takashi; Shoda, Makoto

doi:10.1007/bf02447729

Cited by 3 publications

(2 citation statements)

References 17 publications

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“…DNA was separated on 0.7% agarose gels containing 0.5 mg mL À1 ethidium bromide at 85 V for 120 min. Band intensity was evaluated densitometrically using digital image analysis software (LabWorks v. 4.0.0.8; Upland, CA), using the method of Asaka et al (1994) with minor modifications. Briefly, DNA concentrations were determined using a 2-10 kbp supercoiled plasmid marker containing 30 ng DNA mL À1 per band, which served as both a molecular weight marker and an internal standard for normalizing DNA concentrations between gels (Promega, Madison, WI).…”

Section: Plasmid Analysismentioning

confidence: 99%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.

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Section: Plasmid Analysismentioning

confidence: 99%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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“…This may be due however, to the difference in copy numbers of pSB2019 replicated in E. coli versus L. innocua. Plasmid SB2019 is a derivative of pMK4 which is a shuttle vector and replicates in E. coli in high copy numbers [22] but only approximately 15 copies per cell in Bacillus subtilis [44][45][46]. In a study of optimization of gfp expression vectors for detection of L. monocytogenes, Fortineau et al [47] also observed relatively low fluorescence intensity emitted by L. monocytogenes cells transformed with integrative monocopy and low copy gfp containing plasmids.…”

Section: Discussionmentioning

confidence: 93%

Autofluorescence and green fluorescent protein-derived fluorescence in Listeria innocua

Friedly

Chalova

Crandall

et al. 2007

Sens. & Instrumen. Food Qual.

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Fluorescent L. innocua M1 was generated by transformation with plasmid SB2019 carrying gfp3. The transformed organism exhibited 2 h longer lag phase than the parental strain. The transformation and gfp3 expression did not affect the growth rates (0.49 ± 0.02 and 0.47 ± 0.05 h -1 ) and the maximum optical densities (1.02 ± 0.01 and 0.90 ± 0.06) of the parental or the transformed strains. The transformation of L. innocua M1 with pSB2019 resulted in cell-concentration related fluorescence which was detectable from washed cells but not in growth media. Statistical discrimination between GFP3-driven and background fluorescence signals of transformed and parental strains occurred at cell optical densities of 0.1 and above which was partially due to a relatively high endogenous autofluorescence. Although the gfp-transformed L. innocua M1 developed in this study has the potential to be a marker organism for monitoring Listeria spp. responses in mixed cultures, more work is needed to optimize the GFP-based fluorescent signal.

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A Plasmid Isolated from Phytopathogenic Onion Yellows Phytoplasma and Its Heterogeneity in the Pathogenic Phytoplasma Mutant

Kuboyama¹,

Huang²,

Lu³

et al. 1998

MPMI

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A 3.6-kbp DNA fragment was cloned from the extrachromosomal DNA of a pathogenic plant mollicute, onion yellows phytoplasma (OY-W). Sequence analysis of the fragment revealed an open reading frame (ORF) encoding the replication (Rep) protein of rolling-circle replication (RCR)-type plasmids. This result suggests the existence of a plasmid (pOYW1) in OY-W that uses the RCR mechanism. This assumption was confirmed by detecting the single-stranded DNA (ssDNA) of a replication intermediate that is specifically produced by the RCR mechanism. This is the first report on the identification of the replication system of this plasmid and the genes encoded in it. With a DNA fragment including the Rep gene region of pOYW1 used as a probe, Southern and Northern (RNA) blot hybridizations were employed to examine the heterogeneity between the plasmids found in OY-W and a pathogenic mutant (OY-M) isolated from OY-W. Multiple bands were detected in the DNA and RNA extracted from both OY-W and OY-M infected plants, although the banding patterns were different. Moreover, the copy number of plasmids from OY-W was about 4.2 times greater than that from OY-M. These results indicate constructive heterogeneity between OY-W and OY-M plasmids, and the possibility of a relationship between the plasmid-encoded genes and the pathogenicity of the phytoplasma was suggested.

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A rapid and simple method for plasmid copy number comparison

Cited by 3 publications

References 17 publications

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Autofluorescence and green fluorescent protein-derived fluorescence in Listeria innocua

A Plasmid Isolated from Phytopathogenic Onion Yellows Phytoplasma and Its Heterogeneity in the Pathogenic Phytoplasma Mutant

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