Detection Of DNA Damage By Use Of Escherichia Coli Carrying recA\u27::lux, uvrA\u27::lux, And alkA\u27::lux Reporter Plasmids

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor\u27s reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair

Oxidative Stress Detection With Escherichia Coli Harboring A katG\u27::lux Fusion

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG\u27::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards

In Vivo Titration Of Mitomycin C Action By The Presence Of Four Escherichia Coli Genomic Regions On Multicopy Plasmid

Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinct E. coli genes (mdfA, gyrl, rob, and sdiA) on high-copy-number vectors. mdfA encodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division

Bioluminescent Escherichia Coli Strains For The Quantitative Detection Of Phosphate And Ammonia In Coastal And Suburban Watersheds

Accumulation of phosphate and ammonia in estuarine systems and subsequent dinoflagellate and algal blooms has been implicated in fish kills and in health risks for fishermen. Analytic chemistry kits are used to measure phosphate and ammonia levels in water samples, but their sensitivity is limited due to specificity for inorganic forms of these moieties. An Escherichia coli bioluminescent reporter system measured the bioavailability of inorganic nutrients through fusion of E. coli promoters (phoA or glnAp2) to the luxCDABE operon of Vibrio fischeri carried either on the chromosome or on a multicopy plasmid vector, resulting in emission of light in response to phosphate or ammonia starvation. Responses were shown to be under the control of expected physiological regulators, phoB and glnFG, respectively. Standard curves were used to determine the phosphate and ammonia levels in water samples from diverse watersheds located in the northeastern United States. Bioluminescence produced in response to nutrient starvation correlated with concentrations of phosphate (1-24 ppm) and ammonia (0.1-1.6 ppm). While the ammonia biosensor measured nutrient concentrations in tested water samples that were comparable to the amounts reported by a commercial kit, the phosphate biosensor reported higher levels of phosphate in Chesapeake water samples than did the kit

Responses To Toxicants Of An Escherichia Coli Strain Carrying A uspA\u27::lux Genetic Fusion And An E. Coli Strain Carrying A grpE\u27::lux Fusion Are Similar

A transcriptional fusion of the Escherichia coli uspA promoter to luxCDABE was characterized and compared with a heat shock-responsive grpE\u27::lux fusion. Similarities in range and rank order of inducing conditions were observed; however, the magnitude of induction was typically greater for the grpE\u27::lux fusion strain

Development of a prototype superconducting radio-frequency cavity for conduction-cooled accelerators

The higher efficiency of superconducting radio-frequency (SRF) cavities compared to normal-conducting ones enables the development of high-energy continuous-wave linear accelerators (linacs). Recent progress in the development of high-quality Nb$_3$Sn film coatings along with the availability of cryocoolers with high cooling capacity at 4 K makes it feasible to operate SRF cavities cooled by thermal conduction at relevant accelerating gradients for use in accelerators. A possible use of conduction-cooled SRF linacs is for environmental applications, requiring electron beams with energy of $1 - 10$ MeV and 1 MW of power. We have designed a 915 MHz SRF linac for such an application and developed a prototype single-cell cavity to prove the proposed design by operating it with cryocoolers at the accelerating gradient required for 1 MeV energy gain. The cavity has a $\sim 3$ $\mu$m thick Nb$_3$Sn film on the inner surface, deposited on a $\sim4$ mm thick bulk Nb substrate and a bulk $\sim7$ mm thick Cu outer shell with three Cu attachment tabs. The cavity was tested up to a peak surface magnetic field of 53 mT in liquid He at 4.3 K. A horizontal test cryostat was designed and built to test the cavity cooled with three Gifford-McMahon cryocoolers. The rf tests of the conduction-cooled cavity, performed at General Atomics, achieved a peak surface magnetic field of 50 mT and stable operation was possible with up to 18.5 W of rf heat load. The peak frequency shift due to microphonics was 23 Hz. These results represent the highest peak surface magnetic field achieved in a conduction-cooled SRF cavity to date and meet the requirements for a 1 MeV energy gain

High quality RNA isolation from Aedes aegypti midguts using laser microdissection microscopy

Background: Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti

Measurements of Higgs boson production and couplings in diboson final states with the ATLAS detector at the LHC

Measurements are presented of production properties and couplings of the recently discovered Higgs boson using the decays into boson pairs, H →γ γ, H → Z Z∗ →4l and H →W W∗ →lνlν. The results are based on the complete pp collision data sample recorded by the ATLAS experiment at the CERN Large Hadron Collider at centre-of-mass energies of √s = 7 TeV and √s = 8 TeV, corresponding to an integrated luminosity of about 25 fb−1. Evidence for Higgs boson production through vector-boson fusion is reported. Results of combined fits probing Higgs boson couplings to fermions and bosons, as well as anomalous contributions to loop-induced production and decay modes, are presented. All measurements are consistent with expectations for the Standard Model Higgs boson

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011