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 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 Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

2020 ◽  
Vol 117 (28) ◽  
pp. 16537-16545 ◽  
Author(s):  
Nan Xiang ◽  
Chenyue Guo ◽  
Jiwei Liu ◽  
Hao Xu ◽  
Ray Dixon ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Klebsiella Oxytoca ◽  
Plant Mitochondria ◽  
Eukaryotic Cells ◽  
Mofe Protein ◽  
The Stability ◽  
Biological Nitrogen ◽  
Nif Proteins

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction ofnifgenes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein fromAzotobacter vinelandiiandKlebsiella oxytocawere found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, fromK. oxytocathat confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast,Oryza sativa,Nicotiana tabacum, andArabidopsis thalianainEscherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

scholarly journals Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes

2009 ◽  
Vol 191 (14) ◽  
pp. 4534-4545 ◽  
Author(s):  
João C. Setubal ◽  
Patricia dos Santos ◽  
Barry S. Goldman ◽  
Helga Ertesvåg ◽  
Guadelupe Espin ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Formate Dehydrogenase ◽  
Azotobacter Vinelandii ◽  
Chemical Agents ◽  
Oxygen Sensitive ◽  
Obligate Aerobe ◽  
Obligate Aerobic ◽  
Respiratory Proteins ◽  
Physical And Chemical

ABSTRACT Azotobacter vinelandii is a soil bacterium related to the Pseudomonas genus that fixes nitrogen under aerobic conditions while simultaneously protecting nitrogenase from oxygen damage. In response to carbon availability, this organism undergoes a simple differentiation process to form cysts that are resistant to drought and other physical and chemical agents. Here we report the complete genome sequence of A. vinelandii DJ, which has a single circular genome of 5,365,318 bp. In order to reconcile an obligate aerobic lifestyle with exquisitely oxygen-sensitive processes, A. vinelandii is specialized in terms of its complement of respiratory proteins. It is able to produce alginate, a polymer that further protects the organism from excess exogenous oxygen, and it has multiple duplications of alginate modification genes, which may alter alginate composition in response to oxygen availability. The genome analysis identified the chromosomal locations of the genes coding for the three known oxygen-sensitive nitrogenases, as well as genes coding for other oxygen-sensitive enzymes, such as carbon monoxide dehydrogenase and formate dehydrogenase. These findings offer new prospects for the wider application of A. vinelandii as a host for the production and characterization of oxygen-sensitive proteins.

scholarly journals Inactivation of the ampDE Operon Increases Transcription of algD and Affects Morphology and Encystment of Azotobacter vinelandii

2000 ◽  
Vol 182 (17) ◽  
pp. 4829-4835 ◽  
Author(s):  
Cinthia Núñez ◽  
Soledad Moreno ◽  
Luis Cárdenas ◽  
Gloria Soberón-Chávez ◽  
Guadalupe Espín
Keyword(s):  
Escherichia Coli ◽  
Biosynthetic Pathway ◽  
Azotobacter Vinelandii ◽  
Transmembrane Protein ◽  
Nucleotide Sequencing ◽  
Insertion Mutant ◽  
Gene Encoding ◽  
Alginate Production ◽  
Key Enzyme

ABSTRACT Transcription of algD, encoding GDP-mannose dehydrogenase, the key enzyme in the alginate biosynthetic pathway, is highly regulated in Azotobacter vinelandii. We describe here the characterization of a Tn5 insertion mutant (AC28) which shows a higher level of expression of analgD::lacZ fusion. AC28 cells were morphologically abnormal and unable to encyst. The cloning and nucleotide sequencing of the Tn5-disrupted locus in AC28 revealed an operon homologous to the Escherichia coli ampDEoperon. Tn5 was located within the ampD gene, encoding a cytosolicN-acetyl-anhydromuramyl-l-alanine amidase that participates in the intracellular recycling of peptidoglycan fragments. The ampE gene encodes a transmembrane protein, but the function of the protein is not known. We constructed strains carryingampD or ampE mutations and one with anampDE deletion. The strain with a deletion of theampDE operon showed a phenotype similar to that of mutant AC28. The present work demonstrates that both alginate production and bacterial encystment are greatly influenced by the bacterial ability to recycle its cell wall.

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 Key Challenges Facing AMISOM in Military Diplomacy in the Horn of Africa

2021 ◽  
Vol 2 (2) ◽  
pp. 54-67
Author(s):  
Musoma Albert Lusiola
Keyword(s):  
Data Analysis ◽  
Mixed Methods Research ◽  
Strategic Alliance ◽  
Primary Data ◽  
Peace Operations ◽  
Horn Of Africa ◽  
Design Data ◽  
Competing Interests ◽  
Mission Teams ◽  
Military Diplomacy

The world over, military diplomacy has not been always successful. This stems out of the fact that it could be faced by a plethora of challenges. This paper sets out to explore the key challenges facing AMISOM in military diplomacy in the Horn of Africa.  The study adopted an exploratory and mixed methods research design. Data was obtained from a sample of 100 persons sampled from a population of 22,315 AMISOM Staff and Civilian contingent. The study employed a breadth of both primary and secondary sources for data collection. Primary data was being collected from study respondents by means of a research questionnaire and an interview schedule. The data analysis process involved both qualitative and quantitative techniques. Content analysis was mainly used to analyze the qualitative data and which would be reported normatively. Quantitative research findings were analyzed and reported using descriptive statistics, tables, graphs, charts and inferential statistics in Statistical Package for Social Sciences (SPSS v23). Moreover, the data analysis was structured objectively to address each of the study research questions.  The findings show that while competing interests may have clouded the scene at regional level, partly alluded to lack of a common approach to deal with the Somalia issue among the various countries, military diplomatic engagements by AMISOM are the most agreeable way to address regional peace and security. The study highlights the greater need for revised strategies in military diplomacy efforts and novel approaches to address competing interests among troop contributing countries that comprise AMISOM. Based on the study findings, the following recommendations were made. Arguably, the most important dimension of its success is hinged on the strategic unity and partnership of the different troops. At present however, the inconsistency in unity and strategic alliance among these countries continue to challenge the seamless command and probably influence the implementation of different military diplomacy strategies based on competing interests. Further, while assets remain a critical component of military diplomacy, the success of such multidimensional peace operations is equally anchored on a civilian component and the need for civilian capabilities. The realization of effective peacemaking and peacekeeping calls for efficient management structures at the field and in Addis for strategic and support of mission teams. AMISOM currently experiences an insufficient institutional capacity and human resources required to effectively handle complex peace operations and peacemaking initiatives. Recent assessment reveals the institution bureaucratic processes are still weak.

Enhancing digital design data availability in the aerospace industry

2010 ◽  
Vol 2 (4) ◽  
pp. 240-246 ◽  
Author(s):  
E. Shehab ◽  
M. Bouin-Portet ◽  
R. Hole ◽  
C. Fowler
Keyword(s):  
Aerospace Industry ◽  
Digital Design ◽  
Data Availability ◽  
Design Data
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