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Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9

Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9 tadalafil generico

Abstract

Background
Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods. viagra generic

Results
In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h. cialis coupon 2017

Conclusion
In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci. cialis 20mg prix en pharmacie

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Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Easy regulation of metabolic flux in Escherichia coli using an endogenous type I-E CRISPR-Cas system

Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a recently developed powerful tool for gene regulation. In Escherichia coli, the type I CRISPR system expressed endogenously shall be easy for internal regulation without causing metabolic burden in compared with the widely used type II system, which expressed dCas9 as an additional plasmid.

 

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The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagatella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13 A). The maximum specific growth rate increased from 0.13 up to 0.18 h−1and a biomass yield coefficient of 0.56 gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker’s yeast.

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Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A Review

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A Review

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO2, H2O and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. This article is protected by copyright. All rights reserved.

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Functional Membrane Microdomains Organize Signaling Networks in Bacteria

Functional Membrane Microdomains Organize Signaling Networks in Bacteria

Membrane organization is usually associated with the correct function of a number of cellular processes in eukaryotic cells as diverse as signal transduction, protein sorting, membrane trafficking, or pathogen invasion. It has been recently discovered that bacterial membranes are able to compartmentalize their signal transduction pathways in functional membrane microdomains (FMMs). In this review article, we discuss the biological significance of the existence of FMMs in bacteria and comment on possible beneficial roles that FMMs play on the harbored signal transduction cascades. Moreover, four different membrane-associated signal transduction cascades whose functions are linked to the integrity of FMMs are introduced, and the specific role that FMMs play in stabilizing and promoting interactions of their signaling components is discussed. Altogether, FMMs seem to play a relevant role in promoting more efficient activation of signal transduction cascades in bacterial cells and show that bacteria are more sophisticated organisms than previously appreciated.

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US researchers develop new ways of producing biofuels from E-coli

US researchers develop new ways of producing biofuels from E-coli
US researchers from Washington University have developed a new way to harness the chemical production properties of E.coli bacteria, making the production of certain biofuels from the bacteria more efficient. The new method, described in a paper published in the journal Metabolic Engineering, involves the development of two different protein pathways capable of chemically affecting the production of biofuel in E.coli. Previously, researchers have had difficulty producing branched-chain fatty acids (BCFA) as common bacteria like E.coli mostly produce straight-chain fatty acids (SCFA), which have inferior fuel properties. The new method could enable the E.coli bacteria to boost its BCFA production to 80% of all fuel products.

Synthetic Genomics team engineers Vmax™, an advantaged next-generation host organism for a wide range of biotechnology applications

Synthetic Genomics team engineers Vmax™, an advantaged next-generation host organism for a wide range of biotechnology applications

Optimized system has potential to replace the workhorse E. coli by increasing speed and efficiency of protein production and cloning

Researchers from Synthetic Genomics, Inc. (SGI) announced today the development and extensive engineering of Vibrio natriegens into a next-generation biotechnology host organism Vmax™. Looking to accelerate the pace of discovery and the path to sustainable solutions, the team set out to develop a novel bacterial host that will drastically reduce the amount of time scientists spend on each experiment and workflow and to enhance productivity of the resulting new host.

After screening for the fastest-growing strain and optimizing methods for introducing DNA into those cells at high efficiencies, the team developed genome engineering tools to improve the performance of Vmax™ for common biotech applications, namely, recombinant protein expression and molecular cloning. These breakthroughs build on expertise gleaned during the creation of the first synthetic cell and first minimal cell and again position SGI at the forefront of synthetic biology.

The paper describing this work is the first peer-reviewed publication of its kind and was published online today in Nature Methods by Matthew T. Weinstock, Eric D. Hesek, Christopher M. Wilson, and Daniel G. Gibson.

“This work provides a game-changing alternative to E. coli, the organism that has been a laboratory staple for decades, and again highlights the rapid and innovative synthetic biology expertise we’ve developed at SGI. We are in the process of designing and synthesizing new Vmax™ cells that operate at even higher efficiencies and productivity as we move toward a next-generation host for protein production,” said Daniel Gibson, Vice President, DNA Technologies, SGI.

Commenting on the origin of the research, Todd Peterson, Chief Technology Officer at SGI stated, “Despite the known drawbacks and shortcomings, scientists have been necessitated to use E. coli as a laboratory host primarily because there have been no suitable alternatives. We deployed our synthetic biology expertise to develop a new host strain that will drastically improve upon the traditional methods and tools.”

Typical cloning projects using E. coli competent cells span several days starting from the time a cloning process is initiated to the time plasmid DNA is prepared. Cloning strategies employing Vmax™ developed by the SGI team shorten that time to as little as one day.

The advancements described by the team set the stage for commercialization of these next-generation cells for cloning and protein expression by SGI in the coming months. Vmax™ is compatible with most kits, reagents, growth medium, vectors, and procedures already entrenched in laboratories. Making these cells commercially available will accelerate the pace of global biotechnological research, making a far-reaching and lasting impact toward genetic exploration and discovery worldwide.

About Synthetic Genomics Inc.
Synthetic Genomics Inc. (SGI), located in La Jolla, CA, is a leader in the fields of synthetic biology and synthetic genomics, advancing genomics to better life. SGI applies its intellectual property in this rapidly evolving field to design and build biological systems solving global sustainability challenges. SGI serves three end markets: research, bioproduction, and applied products. The company’s research offerings, commercialized through its subsidiary SGI-DNA, are revolutionizing science and medicine with next-generation genomic solutions, including the world’s first DNA printer. SGI applies its integrated synthetic biology capabilities to reinvent bio-based production by improving existing production systems and developing novel, optimized production hosts. SGI develops its applied products, typically in partnership with leading global organizations, across a variety of industries including sustainable bio-fuels, sustainable crops, nutritional supplements, vaccines, and transplantable organs.

About SGI-DNA
SGI-DNA, a wholly owned subsidiary of Synthetic Genomics, Inc (SGI), is responsible for all commercial aspects of SGI’s synthetic DNA business and focuses on strategic business relationships with both academic and commercial researchers. Building on the scientific advancements and breakthroughs from leading scientists such as J. Craig Venter, Ham Smith, Clyde Hutchison, Dan Gibson and their teams, SGI-DNA utilizes unique and proprietary DNA technologies to produce complex synthetic genes and reagents. SGI-DNA also offers the BioXp™ 3200 System, the world’s first DNA printer, in addition to a comprehensive suite of genomic services, including whole genome sequencing, library design, bioinformatics services, and reagent kits to enable synthetic biology.

 

http://www.syntheticgenomics.com

 

Naturally occurring transporter protein discovered which boosts rice yield by 50%

In collaboration with researchers at Nanjing Agricultural University, Dr Tony Miller from the John Innes Centre has developed rice crops with an improved ability to manage their own pH levels, enabling them to take up significantly more nitrogen, iron and phosphorous from soil and increase yield by up to 54%.

Rice is a major crop, feeding almost 50% of the world’s population and has retained the ability to survive in changing environmental conditions. The crop is able to thrive in flooded paddy fields – where the soggy, anaerobic conditions favour the availability of ammonium – as well as in much drier, drained soil, where increased oxygen means more nitrate is available. nitrogen fertilizer is a major cost in growing many cereal crops and its overuse has a negative environmental impact.

The nitrogen that all plants need to grow is typically available in the form of nitrate or ammonium ions in the soil, which are taken up by the plant roots. For the plant, getting the right balance of nitrate and ammonium is very important: too much ammonium and plant cells become alkaline; too much nitrate and they become acidic. Either way, upsetting the pH balance means the plant’s enzymes do not work as well, affecting plant health and crop yield.

Together with the partners in Nanjing, China, Dr Miller’s team has been working out how rice plants can maintain pH under these changing environments.

Rice contains a gene called OsNRT2.3, which creates a protein involved in nitrate transport. This one gene makes two slightly different versions of the protein: OsNRT2.3a and OsNRT2.3b. Following tests to determine the role of both versions of the protein, Dr Miller’s team found that OsNRT2.3b is able to switch nitrate transport on or off, depending on the internal pH of the plant cell.

When this ‘b’ protein was overexpressed in rice plants they were better able to buffer themselves against pH changes in their environment. This enabled them to take up much more nitrogen, as well as more iron and phosphorus. These rice plants gave a much higher yield of rice grain (up to 54% more yield), and their nitrogen use efficiency increased by up to 40%.

Dr Miller said: “Now that we know this particular protein found in rice plants can greatly increase nitrogen efficiency and yields, we can begin to produce new varieties of rice and other crops. These findings bring us a significant step closer to being able to produce more of the world’s food with a lower environmental impact.”

This new technology has been patented by PBL, the John Innes Centre’s innovation management company, and has already been licensed to three different companies to develop new varieties of six different crop species.

This study, which will be published in the Proceedings of the National Academy of Sciences USA, was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and grants from the Chinese Government.

 

The paper “Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields” has been published in the Proceedings of the National Academy of Science www.pnas.org/content/early/2016/06/01/1525184113.full

Full article from BBSRC http://www.bbsrc.ac.uk/news/food-security/2016/160628-pr-protein-discovered-boosts-rice-yield/?utm_source=MailingList&utm_medium=email&utm_campaign=BBSRC+News+-+July+2016

 

Development of an accurate kinetic model for the central carbon metabolism of Escherichia coli

Development of an accurate kinetic model for the central carbon metabolism of Escherichia coli

Background

A kinetic model provides insights into the dynamic response of biological systems and predicts how their complex metabolic and gene regulatory networks generate particular functions. Of many biological systems,Escherichia coli metabolic pathways have been modeled extensively at the enzymatic and genetic levels, but existing models cannot accurately reproduce experimental behaviors in a batch culture, due to the inadequate estimation of a specific cell growth rate and a large number of unmeasured parameters.

Results

In this study, we developed a detailed kinetic model for the central carbon metabolism of E. coli in a batch culture, which includes the glycolytic pathway, tricarboxylic acid cycle, pentose phosphate pathway, Entner-Doudoroff pathway, anaplerotic pathway, glyoxylate shunt, oxidative phosphorylation, phosphotransferase system (Pts), non-Pts and metabolic gene regulations by four protein transcription factors: cAMP receptor, catabolite repressor/activator, pyruvate dehydrogenase complex repressor and isocitrate lyase regulator. The kinetic parameters were estimated by a constrained optimization method on a supercomputer. The model estimated a specific growth rate based on reaction kinetics and accurately reproduced the dynamics of wild-type E. coli and multiple genetic mutants in a batch culture.

Conclusions

This model overcame the intrinsic limitations of existing kinetic models in a batch culture, predicted the effects of multilayer regulations (allosteric effectors and gene expression) on central carbon metabolism and proposed rationally designed fast-growing cells based on understandings of molecular processes.

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Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Membrane engineering via trans unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables

Constructing microbial biocatalysts that produce biorenewables at economically viable yields and titers is often hampered by product toxicity. For production of short chain fatty acids, membrane damage is considered the primary mechanism of toxicity, particularly in regards to membrane integrity. Previous engineering efforts in Escherichia coli to increase membrane integrity, with the goal of increasing fatty acid tolerance and production, have had mixed results. Herein, a novel approach was used to reconstruct the E. coli membrane by enabling production of a novel membrane component. Specifically, trans unsaturated fatty acids (TUFA) were produced and incorporated into the membrane of E. coli MG1655 by expression of cis-trans isomerase (Cti) from Pseudomonas aeruginosa. While the engineered strain was found to have no increase in membrane integrity, a significant decrease in membrane fluidity was observed, meaning that membrane polarization and rigidity were increased by TUFA incorporation. As a result, tolerance to exogenously added octanoic acid and production of octanoic acid were both increased relative to the wild-type strain. This membrane engineering strategy to improve octanoic acid tolerance was found to require fine-tuning of TUFA abundance. Besides improving tolerance and production of carboxylic acids, TUFA production also enabled increased tolerance in E. coli to other bio-products, e.g. alcohols, organic acids, aromatic compounds, a variety of adverse industrial conditions, e.g. low pH, high temperature, and also elevated styrene production, another versatile bio-chemical product. TUFA permitted enhanced growth due to alleviation of bio-product toxicity, demonstrating the general effectiveness of this membrane engineering strategy towards improving strain robustness.

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