scholarly journals Mutagenesis in the lacI gene target of E. coli: Improved analysis for lacId and lacO mutants

2014 ◽  
Vol 770 ◽  
pp. 79-84 ◽  
Author(s):  
Sarah J. Swerdlow ◽  
Roel M. Schaaper
Keyword(s):  
Gene Target ◽  
E Coli ◽  
Laci Gene

scholarly journals RegR Virulence Regulon of Rabbit-Specific Enteropathogenic Escherichia coli Strain E22

2013 ◽  
Vol 81 (4) ◽  
pp. 1078-1089 ◽  
Author(s):  
Yogitha N. Srikhanta ◽  
Dianna M. Hocking ◽  
Judyta Praszkier ◽  
Matthew J. Wakefield ◽  
Roy M. Robins-Browne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Bioinformatic Analysis ◽  
Coli Strain ◽  
Mobility Shift ◽  
Gene Encoding ◽  
Gene Target ◽  
Content Type ◽  
E Coli ◽  
Genes Encoding

ABSTRACTAraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenicEscherichia coli(EPEC), enterotoxigenicE. coli, enteroaggregativeE. coli, andCitrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, ofC. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target,sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression ofsefAby binding to a region upstream of thesefApromoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22.

Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli

1985 ◽  
Vol 201 (1) ◽  
pp. 35-37 ◽  
Author(s):  
Roshan B. Christensen ◽  
J. R. Christensen ◽  
Christopher W. Lawrence
Keyword(s):  
Spontaneous Mutation ◽  
E Coli ◽  
Laci Gene

scholarly journals THE EFFECTS OF CRUDE RECOMBINANT VIRAL PROTEIN VACCINES AGAINST GROUPER SLEEPY DISEASE IRIDOVIRUS (GSDIV) ON HUMPBACK GROUPER (Cromileptes altivelis)

2015 ◽  
Vol 10 (2) ◽  
pp. 163
Author(s):  
Ketut Mahardika ◽  
Indah Mastuti
Keyword(s):  
Capsid Protein ◽  
Expression System ◽  
Survival Rates ◽  
Coli Strain ◽  
Sea Bream ◽  
Recombinant Bacteria ◽  
Gene Target ◽  
E Coli ◽  
Pi Index ◽  
Potential Vaccine

Infection of Megalocytivirus cause serious mass mortality in marine fish in South East Asian countries. The aim of this study was to produce recombinant of GSDIV capsid protein and its protection to humpback grouper Cromileptes altivelis against grouper sleepy disease iridovirus (GSDIV). A major capsid protein (MCP) was selected for use as a crude subunit vaccines. This gene target (MCP) was inserted to the protein expression system vector of pET SUMO and cloned in cells bacteria Escherichia coli strain BL-21. The MCP was succeded to be induced using 1 mM of IPTG. Results of protein analysis using MALDI TOF-TOF indicated that the MCP has measurement of 49.566 kDa with PI index of 6.00, and contained 453 amino acids. BLAST homology analysis exhibited that the amino acid sequence of the MCP showed high similarity with MCP of Red Sea Bream Iridovirus (RSIV). E. coli expressing MCP protein was inactivated using 0.03% formalin overnight and washed using PBS. The inactivated E. coli as a crude subunit vaccine was then injected intramuscularly to humpback grouper juveniles. Subsequently, the juveniles were challenged tested with GSDIV. The juveniles vaccinated with the MCP recombinant bacteria showed significantly higher survival rates than control those vaccinated with PBS. Thus, the MCP fusion protein is considered as a potential vaccine against GSDIV infections in grouper.

scholarly journals DNA SEQUENCE ANALYSIS OF MUTAGENICITY AND SITE SPECIFICITY OF ETHYL METHANESULFONATE IN Uvr+ AND UvrB- STRAINS OF ESCHERICHIA COLI

Genetics ◽  
1986 ◽  
Vol 113 (4) ◽  
pp. 811-819
Author(s):  
Philip A Burns ◽  
Frances L Allen ◽  
Barry W Glickman
Keyword(s):  
Dna Sequence ◽  
Excision Repair ◽  
Base Sequence ◽  
Site Specificity ◽  
Repair System ◽  
Induced Mutations ◽  
Base Pairs ◽  
E Coli ◽  
Laci Gene ◽  
Induced Mutagenesis

ABSTRACT EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strians; G:C → A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed pre-mutagenic lesion, O  6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O  6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.

scholarly journals I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins

2020 ◽  
Vol 48 (5) ◽  
pp. e28-e28
Author(s):  
Sarolta Szentes ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Eszter Zsigmond ◽  
Pál Salamon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Binding ◽  
Lac Repressor ◽  
Host Bacterium ◽  
Lambda Phage ◽  
Simple Method ◽  
E Coli ◽  
Laci Gene ◽  
Compatible Plasmid ◽  
Nhei Site

Abstract We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

scholarly journals Evaluation and comparison of recombinase polymerase amplification coupled with lateral-flow bioassay for Escherichia coli O157:H7 detection using diifeerent genes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alka Rani ◽  
Vivek B. Ravindran ◽  
Aravind Surapaneni ◽  
Esmaeil Shahsavari ◽  
Nagalakshmi Haleyur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Professional Training ◽  
Gene Selection ◽  
Point Of Care ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Gene Target ◽  
Microbial Detection ◽  
E Coli ◽  
Simple Detection

AbstractShiga toxin-producing Escherichia coli serotype O157:H7 is a food and waterborne zoonotic pathogen causing gastroenteritis in humans. Rapid and simple detection in water and food is imperative to control its spread. However, traditional microbial detection approaches are time-consuming, expensive and complex to operate at the point-of-care without professional training. We present a rapid, simple, sensitive, specific and portable method for detection of E. coli O157:H7 in drinking water, apple juice and milk. We evaluated the effect of gene selection in detecting E. coli O157:H7 using recombinase polymerase amplification coupled with a lateral flow assay using rfbE, fliC and stx gene targets. As low as 100 ag and 1 fg DNA, 4–5 CFU/mL and 101 CFU/mL of E. coli O157:H7 was detected using the stx and rfbE gene targets respectively with 100% specificity, whilst the detection limit was 10 fg DNA and 102 CFU/mL for the fliC gene target, with 72.8% specificity. The RPA-LFA can be completed within 8 min at temperatures between 37 and 42 °C with reduced handling and simple equipment requirements. The test threshold amplification of the target was achieved in 5–30 min of incubation. In conclusion, RPA-LFA represents a potential rapid and effective alternative to conventional methods for the monitoring of E. coli O157:H7 in food and water.

Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

1974 ◽  
Vol 32 ◽  
pp. 148-149
Author(s):  
D. E. Philpott ◽  
A. Takahashi
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cells ◽  
Salivary Gland ◽  
Carotid Artery ◽  
Basement Membrane ◽  
Coronary Vessels ◽  
Ruthenium Red ◽  
E Coli ◽  
Old Rats ◽  
The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

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