scholarly journals Targeted Chromosomal Knockouts in Mycoplasma pneumoniae

2010 ◽  
Vol 76 (15) ◽  
pp. 5297-5299 ◽  
Author(s):  
Radha Krishnakumar ◽  
Nacyra Assad-Garcia ◽  
Gwynedd A. Benders ◽  
Quang Phan ◽  
Michael G. Montague ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Mycoplasma Pneumoniae ◽  
Target Gene ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Reading Frame ◽  
Gene Knockouts ◽  
Selection For ◽  
Complete Deletion ◽  
Double Crossovers

ABSTRACT Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.

scholarly journals SynMyco transposon: engineering transposon vectors for efficient transformation of minimal genomes

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 327-339 ◽  
Author(s):  
Ariadna Montero-Blay ◽  
Samuel Miravet-Verde ◽  
Maria Lluch-Senar ◽  
Carlos Piñero-Lambea ◽  
Luis Serrano
Keyword(s):  
Antibiotic Resistance ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Mycoplasma Pneumoniae ◽  
Mycoplasma Gallisepticum ◽  
Model Organisms ◽  
Mycoplasma Species ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Reference Tool

Abstract Mycoplasmas are important model organisms for Systems and Synthetic Biology, and are pathogenic to a wide variety of species. Despite their relevance, many of the tools established for genome editing in other microorganisms are not available for Mycoplasmas. The Tn4001 transposon is the reference tool to work with these bacteria, but the transformation efficiencies (TEs) reported for the different species vary substantially. Here, we explore the mechanisms underlying these differences in four Mycoplasma species, Mycoplasma agalactiae, Mycoplasma feriruminatoris, Mycoplasma gallisepticum and Mycoplasma pneumoniae, selected for being representative members of each cluster of the Mycoplasma genus. We found that regulatory regions (RRs) driving the expression of the transposase and the antibiotic resistance marker have a major impact on the TEs. We then designed a synthetic RR termed SynMyco RR to control the expression of the key transposon vector elements. Using this synthetic RR, we were able to increase the TE for M. gallisepticum, M. feriruminatoris and M. agalactiae by 30-, 980- and 1036-fold, respectively. Finally, to illustrate the potential of this new transposon, we performed the first essentiality study in M. agalactiae, basing our study on more than 199,000 genome insertions.

scholarly journals Fate of Escherichia coli during Ensiling of Wheat and Corn

2005 ◽  
Vol 71 (9) ◽  
pp. 5163-5170 ◽  
Author(s):  
Y. Chen ◽  
S. Sela ◽  
M. Gamburg ◽  
R. Pinto ◽  
Z. G. Weinberg
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Dry Matter ◽  
Fluorescent Protein ◽  
Rapid Decrease ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
E Coli ◽  
Aerobic Stability ◽  
Green Fluorescent

ABSTRACT A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 × 108 CFU/g into direct-cut wheat (348 g of dry matter kg−1), wilted wheat (450 g of dry matter kg−1), and corn (375 g of dry matter kg−1). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.

scholarly journals New Generation of Plasmid Backbones Devoid of Antibiotic Resistance Marker for Gene Therapy Trials

2011 ◽  
Vol 19 (11) ◽  
pp. 1942-1949 ◽  
Author(s):  
Gaëlle Vandermeulen ◽  
Corinne Marie ◽  
Daniel Scherman ◽  
Véronique Préat
Keyword(s):  
Gene Therapy ◽  
Antibiotic Resistance ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
New Generation

scholarly journals Transduction by φBB-1, a Bacteriophage ofBorrelia burgdorferi

2001 ◽  
Vol 183 (16) ◽  
pp. 4771-4778 ◽  
Author(s):  
Christian H. Eggers ◽  
Betsy J. Kimmel ◽  
James L. Bono ◽  
Abdallah F. Elias ◽  
Patricia Rosa ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Direct Evidence ◽  
Kanamycin Resistance ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker ◽  
Kanamycin Resistance Cassette ◽  
Phage Dna ◽  
Disease Agent ◽  
Lyme Disease Agent

ABSTRACT We previously described a bacteriophage of the Lyme disease agentBorrelia burgdorferi designated φBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genusBorrelia. To demonstrate the ability of φBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by φBB-1 released from this B. burgdorferi strain. φBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.

Recommended Assay for Treatment of Chicks to Prevent Salmonella Colonization by ‘Competitive Exclusion’

1989 ◽  
Vol 52 (7) ◽  
pp. 500-502 ◽  
Author(s):  
GEOFFREY C. MEAD ◽  
PAUL A. BARROW ◽  
MICHAEL H. HINTON ◽  
FLORENCE HUMBERT ◽  
CLIVE S. IMPEY ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Competitive Exclusion ◽  
Intestinal Colonization ◽  
Control Groups ◽  
Resistance Marker ◽  
Protection Factor ◽  
Antibiotic Resistance Marker ◽  
And Control ◽  
Live Culture

An assay is described for evaluating live-culture treatment material that may be given orally to chicks to prevent intestinal colonization by non-host-specific salmonellae. Both pre-treated and control chicks are challenged with ca 104 salmonellae/chick, using a strain bearing an antibiotic resistance marker. Chicks are examined 5 d after challenge to determine both the proportion of positive birds in treated and control groups and the levels of Salmonella in the caeca of infected individuals. The efficacy of the treatment is determined by calculation of values for Infection Factor and Protection Factor.

Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria

2015 ◽  
Vol 206 ◽  
pp. 342-351 ◽  
Author(s):  
Markus Woegerbauer ◽  
Josef Zeinzinger ◽  
Richard Alexander Gottsberger ◽  
Kathrin Pascher ◽  
Peter Hufnagl ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Agricultural Soils ◽  
Environmental Pollutants ◽  
Marker Genes ◽  
Resistance Marker ◽  
Antibiotic Resistance Marker
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