scholarly journals Quantification of DNA samples by Ethidium Bromide Spot Technique

2018 ◽  
Author(s):  
Jessica Zhao ◽  
Julian Zhao ◽  
Antoine Cummins ◽  
Tiffany Gonzalez ◽  
Grace Axler-DiPerte ◽  
...  
Keyword(s):  
Ethidium Bromide ◽  
Uv Light ◽  
Spot Size ◽  
Data Availability ◽  
Supplementary Information ◽  
Design Data ◽  
Dna Concentration ◽  
Standard Curve ◽  
Competing Interests ◽  
Concentration Method

AbstractAccurate and quick determination of DNA concentration is critical for the assembly of synthetic constructs, as well as a multitude of other experiments. We sought to optimize an under-utilized and inexpensive approach for determining DNA concentration: a spotting technique that uses the intercalating dye Ethidium Bromide. This technique does not require specialized equipment such as a spectrophotometer, but instead relies on visualization of dye-DNA complex fluorescence when excited by UV light. We modelled and tested a range of parameters for dye concentration and spot size, finding that 15uL spots with 1.0ug/mL Ethidium Bromide produced the most reliable standard curve. More importantly, we hope that our approach can help other labs optimize this protocol for their own experimental setup. Adoption of this technique may help enable development of iGEM teams in resource limited environments and laboratories which do not or cannot employ a satisfactory method for determining DNA concentration.Financial DisclosureWe obtained funding support from Canon Solutions America, and indirect support for this work through the CUNY Research Scholars Program. However, the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors have declared that no competing interests exist.Ethics StatementN/AData AvailabilityYes – all data are fully available without restriction. The data can be found in the associated supplementary materials.Article TagsDNA, Ethidium Bromide, EtBr, Concentration, Method, UV, Gibson AssemblyHeader StatementThis work was assessed during the iGEM/PLOS Realtime Peer Review Jamboree on 23rd February 2018 and has been revised in response to the reviewers. Responses to Reviewers, and supplementary information (including Fig. S1) are available as separate files.

scholarly journals Recombinant expression of Proteorhodopsin and biofilm regulators in Escherichia coli for nanoparticle binding and removal in a wastewater treatment model

2018 ◽  
Author(s):  
Justin Yang ◽  
Yvonne Wei ◽  
Catherine Yeh ◽  
Florence Liou ◽  
William Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wastewater Treatment ◽  
Recombinant Expression ◽  
High Surface ◽  
Volume Ratio ◽  
Data Availability ◽  
Supplementary Information ◽  
Design Data ◽  
Aquatic Life ◽  
Competing Interests

AbstractThe small size of nanoparticles is both an advantage and a problem. Their high surface-area-to-volume ratio enables novel medical, industrial, and commercial applications. However, their small size also allows them to evade conventional filtration during water treatment, posing health risks to humans, plants, and aquatic life. This project aims to remove nanoparticles during wastewater treatment using genetically modified Escherichia coli in two ways: 1) binding citrate-capped nanoparticles with the membrane protein Proteorhodopsin, and 2) trapping nanoparticles using Escherichia coli biofilm produced by overexpressing two regulators: OmpR234 and CsgD. We demonstrate experimentally that Escherichia coli expressing Proteorhodopsin binds to 60 nm citrate-capped silver nanoparticles. We also successfully upregulate biofilm production and show that Escherichia coli biofilms are able to trap 30 nm gold particles. Finally, both Proteorhodopsin and biofilm approaches are able to bind and remove nanoparticles in simulated wastewater treatment tanks. We envision integrating our trapping system in both rural and urban wastewater treatment plants to efficiently capture all nanoparticles before treated water is released into the environment.Financial DisclosureThis work was funded by the Taipei American School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors have declared that no competing interests exist.Ethics StatementN/AData AvailabilityYes – all data are fully available without restriction. Sequences for the plasmids used in this study are available through the Registry of Standard Biological Parts. Links to raw data are included in Supplementary Information.

scholarly journals Recombinant Expression of Aldehyde Dehydrogenase 2 (ALDH2) inEscherichia coliNissle 1917 for Oral Delivery in ALDH2-Deficient Individuals

2019 ◽  
Author(s):  
Tim Ho ◽  
Catherine Chang ◽  
Justin Wu ◽  
Iris Huang ◽  
Leona Tsai ◽  
...  
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Oral Delivery ◽  
Recombinant Expression ◽  
Data Availability ◽  
Supplementary Information ◽  
Aldehyde Dehydrogenase 2 ◽  
Design Data ◽  
E Coli ◽  
Competing Interests ◽  
Oral Conditions

AbstractTurning red after consuming alcohol may seem like a mere social inconvenience. Yet, this flushing response is caused by an accumulation of acetaldehyde, a carcinogenic intermediate of alcohol metabolism. Aldehyde dehydrogenase 2 (ALDH2) deficiency, the result of a point mutation, produces a less efficient ALDH2. The resulting accumulation of acetaldehyde greatly increases the risk of developing esophageal and head and neck cancers. In this study, we produced recombinant ALDH2 in the probioticE. coliNissle 1917, which successfully reduces acetaldehyde levels in simulated oral conditions. Packaged in a hard candy, the ALDH2-probiotic would remain in the mouth to specifically target salivary acetaldehyde. Using mathematical modeling, we also determined how much recombinant ALDH2 is needed to reduce elevated acetaldehyde levels.Financial DisclosureThis work was funded by Taipei American School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors have declared that no competing interests exist.Ethics StatementN/AData AvailabilityYes – all data are fully available without restriction. Sequences for the plasmids used in this study are available through the Registry of Standard Biological Parts. Links to raw data are included in Supplementary Information.

scholarly journals Characterization ofAzotobacter vinelandiiand Kits for Its Synthetic Biology Applications

2017 ◽  
Author(s):  
King Pong Leung ◽  
Jacky Fong Chuen Loo ◽  
Leo Chi U Seak ◽  
Tung Faat Lai ◽  
Kevin Yuk Lap Yip ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Azotobacter Vinelandii ◽  
Transmembrane Protein ◽  
Data Availability ◽  
Aerobic Bacterium ◽  
Design Data ◽  
Competing Interests ◽  
Enzymatic Cascades ◽  
Oxygen Sensitive ◽  
Anaerobic Environment

AbstractAzotobacter vinelandii, a Gram-negative aerobic bacterium with an intracellular anaerobic environment that maintains the oxygen-sensitive enzymatic cascades for nitrogen fixation, could be used to express oxygen-sensitive proteins. However, little is known about the properties ofA. vinelandiifor synthetic biology applications. We therefore first characterized and optimized the conditions for growing and screening BioBrick constructs inA. vinelandiiin the presence of 2 antibiotics, ampicillin and chloramphenicol, and then developed two sets of BioBricks for regulated protein expression. The first kit used T7 RNA polymerase, whose expression is under the control of a nitrogen-repressiblenifHpromoter. The commonly used T7-dependent system inEscherichia colican then be used inA. vinelandii. Because its intracellular anaerobic environment is favorable for processes such as magnetosome biogenesis, we attempted to migrate the biogenesis machineries from the magnetotactic bacteriumMagnetospirillum gryphiswaldensetoA. vinelandii. During this undertaking, another insertion kit construct was developed to allow protein conjugation onto magnetosomes. The kit consists ofmamC, a gene encoding a transmembrane protein on magnetosomes, and multiple restriction sites downstream ofmamCfor fusing a gene of interest. This insertion kit allows the attachment of any desired protein onto the magnetosome membrane by fusing with the mamC protein. We demonstrated the function of this kit by fusing mamC to a GFP nanobody. This kit will facilitate the conjugation of any target protein onto magnetosomes for downstream applications in the future.Financial DisclosureWe received sponsorship from the 2012–15 Teaching Development Grants Triennium, Faculty of Engineering and Biochemistry Program, School of Life Sciences, The Chinese University of Hong Kong. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing InterestsThe authors declare that no competing interests exist.Ethics StatementN/A.Data AvailabilityAll data are fully available without restriction.

scholarly journals Quality assessment of linked Canadian clinical administrative hospital and vital statistics death data

2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Nancy Rodrigues ◽  
Maureen Kelly ◽  
Tobi Henderson
Keyword(s):  
Assessment Tool ◽  
Vital Statistics ◽  
Data Availability ◽  
Supplementary Information ◽  
Five Dimensions ◽  
Tracking Process ◽  
Data Assessment ◽  
Hospital Deaths ◽  
And Performance ◽  
Discharge From Hospital

IntroductionThree Canadian clinical-administrative hospital databases were linked to the Canadian Vital Statistics Death Database (CVSD) to provide information about patients who died following discharge from hospital as well as supplementary information about patients that died in-hospital. Quality was assessed using a guided approach and through feedback from initial users. Objectives and ApproachThe linked datasets were created to develop and validate health care indicators and performance measures and perform outcome analyses. It is therefore imperative to evaluate the data’s fitness for use. Quality was assessed by calculating coverage of deaths for all linked contributors, creating a profile of the linked dataset and analyzing issues that were identified by users. These analyses were guided by an existing Data Source Assessment Tool, which provides a set of criteria that allow for assessment across five dimensions of quality, thus allowing for appropriate determination of a given set of data’s fitness for use. ResultsDeterministic linkage of the datasets resulted in linkage rates that ranged from 66.9% to 90.9% depending on the dataset or data year. Linkage rates also varied by Canadian jurisdictions and patient cohort. Variables had good data availability with rates of 95% or higher. Initial users identified a significant number of duplicate records that were flagged to and corrected by the data supplier. 1.4\% of acute hospital deaths had discrepancies in the death date captured in the two linked sources; the vast majority had a difference of only one day. A user group and issue tracking process were created to share information about the linked data and guarantee that issues are triaged to the appropriate party and allow for timely follow up with the data supplier. Conclusion/ImplicationsDocumentation provided by the data supplier was vital to understanding the linkage methodology and its impact on linkage rates. A guided data assessment ensured that strengths and limitations were identified and shared to support appropriate use. Feedback to the data supplier is supporting ongoing improvements to the linkage methodology.

UV Laser Bonding of Optical Devices on Polymers

2008 ◽  
Vol 580-582 ◽  
pp. 459-462 ◽  
Author(s):  
Joo Han Kim ◽  
Hyang Tae Kim ◽  
Chul Ku Lee
Keyword(s):  
Optical Fibers ◽  
Uv Light ◽  
Uv Curing ◽  
Spot Size ◽  
High Viscosity ◽  
Optical Devices ◽  
Optical Systems ◽  
Bonding Layer ◽  
Low Viscosity ◽  
Laser Bonding

UV curing adhesives have been introduced for bonding various materials at a room temperature. It has the advantage of putting minimum thermal load on the system; however, it is not suitable for precision bonding of micro systems such as micro optical devices because of its high viscosity and poor control of the UV light source. In the present work, a laser-curing bonding process of micro optical devices with a low-viscosity UV polymer adhesive has been developed. A focused Nd:YVO4 laser beam with a spot size of 30 µm with a laser power of 100 ~ 700 mW is used for curing a UV adhesive locally. A thin bonding layer with a thickness of a few hundred nanometers without any thermal effects can be obtained for precision laser bonding for optical fibers. Experimental results are provided and the process characteristics have been discussed. Moreover, potential applications in the field of micro optical systems are introduced as well.

scholarly journals Isolation of Ethidium Bromide Degrading Bacteria from Jharkhand

2017 ◽  
Vol 5 (3) ◽  
pp. 293-301 ◽  
Author(s):  
Anil Kumar ◽  
Preeti Swarupa ◽  
Vikram Pal Gandhi ◽  
Snehal Kumari
Keyword(s):  
Microbial Community ◽  
Ethidium Bromide ◽  
Gel Electrophoresis ◽  
Uv Light ◽  
Liquid Waste ◽  
Agarose Gel ◽  
Degrading Bacteria ◽  
Zone Characteristic ◽  
Waste Soil ◽  
Halo Zone

Ethidium bromide (EtBr) is a carcinogenic and mutagenic agent which is widely used in research laboratories to probe nucleic acids by gel electrophoresis. It is generally buried underground (for solid waste) or disposed of pouring it down the sink (in case of liquid waste). Soil or drain microbial community may be able to take care of such substance else it will lead to contamination of our underground resources or others through defined and undefined routes. In view of the above assumption and literature reports the present study was undertaken to isolate and evaluate bacteria for removal, by bioaccumulation and /or biotransformation, of EtBr from contaminated sources and wastes, before their disposal to the environment. Two distinct bacteria both motile BR3 and BR4 could be identified from agarose-gel-waste containing 0.5-1.0 µg/ml ethidium bromide. Both bacteria were found to grow on EtBr-NA plate (Nutrient-Agar supplemented with EtBr at a concentration of 30 µg/ml) however only BR3 isolate showed large non-fluorescent-halo zone (characteristic to degradation of EtBr) when exposed to trans-UV light. Other isolate BR4 could accumulate EtBr within the colony biomass but did not showed clear (non-fluorescent) hallow zone around it. However the bacterium is not able to utilize the EtBr as a sole carbon source.Int. J. Appl. Sci. Biotechnol. Vol 5(3): 293-301

scholarly journals Data Sets Replicas Placements Strategy from Cost-Effective View in the Cloud

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Xiuguo Wu
Keyword(s):  
System Performance ◽  
Financial Burden ◽  
Storage System ◽  
Cost Effective ◽  
Data Access ◽  
Data Availability ◽  
Data Sets ◽  
Cost Models ◽  
Design Data ◽  
Data Set

Replication technology is commonly used to improve data availability and reduce data access latency in the cloud storage system by providing users with different replicas of the same service. Most current approaches largely focus on system performance improvement, neglecting management cost in deciding replicas number and their store places, which cause great financial burden for cloud users because the cost for replicas storage and consistency maintenance may lead to high overhead with the number of new replicas increased in a pay-as-you-go paradigm. In this paper, towards achieving the approximate minimum data sets management cost benchmark in a practical manner, we propose a replicas placements strategy from cost-effective view with the premise that system performance meets requirements. Firstly, we design data sets management cost models, including storage cost and transfer cost. Secondly, we use the access frequency and the average response time to decide which data set should be replicated. Then, the method of calculating replicas’ number and their store places with minimum management cost is proposed based on location problem graph. Both the theoretical analysis and simulations have shown that the proposed strategy offers the benefits of lower management cost with fewer replicas.

scholarly journals The Generation and Quantification of Resistance to Dimethomorph in Phytophthora infestans

Plant Disease ◽  
2004 ◽  
Vol 88 (9) ◽  
pp. 930-934 ◽  
Author(s):  
J. M. Stein ◽  
W. W. Kirk
Keyword(s):  
Phytophthora Infestans ◽  
Ethidium Bromide ◽  
Uv Light ◽  
Management Strategies ◽  
Resistance Management ◽  
Field Resistance ◽  
Induction Treatment ◽  
Resistance Factors ◽  
Biological Cost ◽  
Leaf Disks

The generation of dimethomorph resistance in Phytophthora infestans was attempted using ethidium bromide/UV light mutagenesis and repeated culturing on dimethomorph-amended medium. Ethidium bromide/UV mutagenesis created two isolates of P. infestans with resistance factors for dimethomorph >20, i.e., the ratio of the 50% effective concentration (EC50) of the mutant to that of the wild-type. With repeated culturing on dimethomorph-amended medium, the rate of growth (mm diameter/day) increased until the tenth subculture for most P. infestans isolates. Resistance factors generated from repeated culturing were <8 for all isolates. For most isolates, the generation of dimethomorph resistance resulted in reduced growth rates on nonamended medium, regardless of the level of resistance or induction treatment. Additionally, the frequency of infection of leaf disks and whole tubers was significantly reduced in >20% of the isolates repeatedly subcultured on dimethomorph-amended medium. Regardless of the induction treatment, reduced fitness was common for all P. infestans isolates, indicating a potential biological cost associated with dimethomorph resistance. Based on these results, the development of field resistance to dimethomorph in P. infestans is unlikely with the currently employed resistance management strategies.

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