The Experts below are selected from a list of 479919 Experts worldwide ranked by ideXlab platform
Osamu Iwata - One of the best experts on this subject based on the ideXlab platform.
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Osamu IwataAbstract:Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-Activated Cell sorting
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Tomoko Abe, Osamu IwataAbstract:Euglena gracilis, a microalgal species of uniCellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-Activated Cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intraCellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis Cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.
Keisuke Goda - One of the best experts on this subject based on the ideXlab platform.
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Osamu IwataAbstract:Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-Activated Cell sorting
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Tomoko Abe, Osamu IwataAbstract:Euglena gracilis, a microalgal species of uniCellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-Activated Cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intraCellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis Cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.
Michael Bott - One of the best experts on this subject based on the ideXlab platform.
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pyruvate carboxylase variants enabling improved lysine production from glucose identified by biosensor based high throughput fluorescence Activated Cell sorting screening
ACS Synthetic Biology, 2019Co-Authors: Maike Kortmann, Christina Mack, Meike Baumgart, Michael BottAbstract:Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in Corynebacterium glutamicum that enable improved l-lysine production from glucose. A suitable reporter strain was transformed with a pyc gene library created by error-prone PCR and screened by fluorescence-Activated Cell sorting (FACS) for Cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration. Two pyruvate carboxylase variants, PCxT343A,I1012S and PCxT132A were identified allowing 9% and 19% increased lysine titers upon plasmid-based expression. Chromosomal expression of PCxT132A and PCxT343A variants led to 6% and 14% higher l-lysine levels. The new PCx variants can be useful also for other microbial strains producing TCA cycle-derived metabolites. Our approach indicates that a biosensor such as pSenLys enables directed evolution of many enzymes involved in converting a carbon source into the target metabolite.
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Pyruvate Carboxylase Variants Enabling Improved Lysine Production from Glucose Identified by Biosensor-Based High-Throughput Fluorescence-Activated Cell Sorting Screening
2019Co-Authors: Maike Kortmann, Christina Mack, Meike Baumgart, Michael BottAbstract:Pyruvate carboxylase is an anaplerotic carbon dioxide-fixing enzyme replenishing the tricarboxylic acid cycle with oxaloacetate during growth on sugars. In this study, we applied a lysine biosensor to identify pyruvate carboxylase variants in Corynebacterium glutamicum that enable improved l-lysine production from glucose. A suitable reporter strain was transformed with a pyc gene library created by error-prone PCR and screened by fluorescence-Activated Cell sorting (FACS) for Cells with increased fluorescence triggered by an elevated cytoplasmic lysine concentration. Two pyruvate carboxylase variants, PCxT343A,I1012S and PCxT132A were identified allowing 9% and 19% increased lysine titers upon plasmid-based expression. Chromosomal expression of PCxT132A and PCxT343A variants led to 6% and 14% higher l-lysine levels. The new PCx variants can be useful also for other microbial strains producing TCA cycle-derived metabolites. Our approach indicates that a biosensor such as pSenLys enables directed evolution of many enzymes involved in converting a carbon source into the target metabolite
Koji Yamada - One of the best experts on this subject based on the ideXlab platform.
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Osamu IwataAbstract:Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-Activated Cell sorting
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efficient selective breeding of live oil rich euglena gracilis with fluorescence Activated Cell sorting
Scientific Reports, 2016Co-Authors: Koji Yamada, Takuto Takeuchi, Sharbanee Mitra, Yusuke Kazama, Keisuke Goda, Kengo Suzuki, Hideyuki Suzuki, Tomoko Abe, Osamu IwataAbstract:Euglena gracilis, a microalgal species of uniCellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions-a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-Activated Cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intraCellular lipids with BODIPY(505/515), and FACS-based isolation of top 0.5% lipid-rich E. gracilis Cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.
Pei-yu Chiou - One of the best experts on this subject based on the ideXlab platform.
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Pulsed laser Activated Cell sorter with dielectrophoretic single stream sheathless focusing
2017 IEEE 30th International Conference on Micro Electro Mechanical Systems (MEMS), 2017Co-Authors: Xiongfeng Zhu, Yu-chun Kung, Michael A. Teitell, Pei-yu ChiouAbstract:We report a novel sheathless microfluidic fluorescence-Activated Cell sorter utilizing size-independent three-dimensional dielectrophoretic (DEP) single stream focusing and pulsed laser Activated Cell sorting (PLACS). This is realized by fabricating a 3D microfluidic device using two glass substrates with patterned electrodes sandwiching a thin and open PDMS channel. DEP forces are provided along a 4cm-long channel that focuses particles into a single stream passing through a fluorescence detection zone regardless of their sizes and types at high flow speeds. Pulsed laser-induced cavitation bubbles are used to provide rapid (∼30 μs) and precise fluid perturbation to sort out particles upon the detection of fluorescence. High purity sorting has been accomplished at a throughput of 1500 particles sec−1.
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pulsed laser Activated Cell sorter placs for high throughput fluorescent mammalian Cell sorting
Proceedings of SPIE, 2014Co-Authors: Yue Chen, Michael A. Teitell, Dino Di Carlo, Aram J Chung, Yu Chung Kung, Pei-yu ChiouAbstract:We present a Pulsed Laser Activated Cell Sorter (PLACS) realized by exciting laser induced cavitation bubbles in a PDMS microfluidic channel to create high speed liquid jets to deflect detected fluorescent samples for high speed sorting. Pulse laser triggered cavitation bubbles can expand in few microseconds and provide a pressure higher than tens of MPa for fluid perturbation near the focused spot. This ultrafast switching mechanism has a complete on-off cycle less than 20 μsec. Two approaches have been utilized to achieve 3D sample focusing in PLACS. One is relying on multilayer PDMS channels to provide 3D hydrodynamic sheath flows. It offers accurate timing control of fast (2 m sec-1) passing particles so that synchronization with laser bubble excitation is possible, an critically important factor for high purity and high throughput sorting. PLACS with 3D hydrodynamic focusing is capable of sorting at 11,000 Cells/sec with >95% purity, and 45,000 Cells/sec with 45% purity using a single channel in a single step. We have also demonstrated 3D focusing using inertial flows in PLACS. This sheathless focusing approach requires 10 times lower initial Cell concentration than that in sheath-based focusing and avoids severe sample dilution from high volume sheath flows. Inertia PLACS is capable of sorting at 10,000 particles sec-1 with >90% sort purity.
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3d pulsed laser triggered high speed microfluidic fluorescence Activated Cell sorter
Analyst, 2013Co-Authors: Yue Chen, Yu-chun Kung, Michael A. Teitell, Pei-yu ChiouAbstract:We report a 3D microfluidic pulsed laser-triggered fluorescence-Activated Cell sorter capable of sorting at a throughput of 23 000 Cells per s with 90% purity in high-purity mode and at a throughput of 45 000 Cells per s with 45% purity in enrichment mode in one stage and in a single channel. This performance is realized by exciting laser-induced cavitation bubbles in a 3D PDMS microfluidic channel to generate high-speed liquid jets that deflect detected fluorescent Cells and particles focused by 3D sheath flows. The ultrafast switching mechanism (20 μs complete on–off cycle), small liquid jet perturbation volume, and three-dimensional sheath flow focusing for accurate timing control of fast (1.5 m s−1) passing Cells and particles are three critical factors enabling high-purity sorting at high-throughput in this sorter.
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Pulsed laser triggered high speed microfluidic fluorescence Activated Cell sorter
2012 IEEE 25th International Conference on Micro Electro Mechanical Systems (MEMS), 2012Co-Authors: Yue Chen, Michael A. Teitell, Dino Di Carlo, Sung Yong Park, Jason S. Hong, Tara Teslaa, Jiang F. Zhong, Pei-yu ChiouAbstract:We report a high speed and high purity pulsed laser triggered fluorescence Activated Cell sorter (PLACS) with a sorting throughput up to 20,000 mammalian Cells/s with 37% sorting purity, 90% Cell viability in enrichment mode, and >90% purity in high purity mode at 1,500 Cells/s or 3,000 beads/s. Fast switching (30 μs) and a small perturbation volume (∼90 pL) is realized by a unique sorting mechanism in which explosive vapor bubbles are generated using focused laser pulses in a single layer microfluidic PDMS channel.
