scholarly journals Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of Corynebacterium glutamicum at oscillating pH values

2021 ◽  
Author(s):  
Sarah Täuber ◽  
Luisa Blöbaum ◽  
Valentin Steier ◽  
Marco Oldiges ◽  
Alexander Grünberger
Keyword(s):  
Single Cell ◽  
Corynebacterium Glutamicum ◽  
Large Scale ◽  
Detailed Comparison ◽  
Proof Of Concept ◽  
Application Range ◽  
Ph Values ◽  
Scale Down ◽  
Ph Conditions

In large-scale bioreactors, gradients in cultivation parameters such as oxygen, substrate and pH result in fluctuating environments. pH fluctuations were identified as a critical parameter for bioprocess performance. Traditionally, scale-down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strain and thus process performance. Here, we demonstrate the application of dynamic microfluidic single-cell cultivation (dMSCC) as a novel scale-down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model parameter. A detailed comparison between two-compartment reactor (two-CR) scale-down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 (~8 min) and disturbed pH 6 (~2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two-CR 27%). Afterward, systematic experiments at different symmetric and asymmetric pH oscillations between pH ranges of 4 −6 and 8 −11 and different intervals from 1 minute to 20 minutes, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions within large-scale bioprocesses, which is difficult to conduct using two-CRs.

scholarly journals Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and emulsion coupling assay as a low-cellularity workflow for single-cell cloning

2020 ◽  
Author(s):  
Tobias Groß ◽  
Csaba Jeney ◽  
Darius Halm ◽  
Günter Finkenzeller ◽  
G. Björn Stark ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Large Scale ◽  
Single Cells ◽  
Protein Detection ◽  
Cell Line Development ◽  
Cell Printing ◽  
Cell Clones ◽  
The University

AbstractThe homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion, creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (surveyor assay), mRNA rtPCR assessing altered gene expression and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing altered bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.Author summaryI completed my undergraduate degree in biochemistry at the University of Ulm and finished my master's degree in pharmaceutical biotechnology at the University of Ulm and University of applied science of Biberach with a focus on biotechnology, toxicology and molecular biology. For my master thesis, I went to the University of Freiburg to the department of microsystems engineering, where I developed a novel workflow for cell line development. I stayed at the institute for my doctorate, but changed my scientific focus to the development of the emulsion coupling technology, which is a powerful tool for the quantitative and highly parallel measurement of protein and protein interactions. I am generally interested in being involved in the development of innovative molecular biological methods that can be used to gain new insights about biological issues. I am particularly curious to unravel the complex and often poorly understood protein interaction pathways that are the cornerstone of understanding cellular functionality and are a fundamental necessity to describe life mechanistically.

scholarly journals Scirpy: A Scanpy extension for analyzing single-cell T-cell receptor sequencing data

2020 ◽  
Author(s):  
Gregor Sturm ◽  
Tamas Szabo ◽  
Georgios Fotakis ◽  
Marlene Haider ◽  
Dietmar Rieder ◽  
...  
Keyword(s):  
T Cell ◽  
Single Cell ◽  
Large Scale ◽  
Single Cells ◽  
Cell Receptor ◽  
Sequencing Data ◽  
Seamless Integration ◽  
T Cell Phenotypes ◽  
Cell Phenotypes

AbstractSummaryAdvances in single-cell technologies have enabled the investigation of T cell phenotypes and repertoires at unprecedented resolution and scale. Bioinformatic methods for the efficient analysis of these large-scale datasets are instrumental for advancing our understanding of adaptive immune responses in cancer, but also in infectious diseases like COVID-19. However, while well-established solutions are accessible for the processing of single-cell transcriptomes, no streamlined pipelines are available for the comprehensive characterization of T cell receptors. Here we propose Scirpy, a scalable Python toolkit that provides simplified access to the analysis and visualization of immune repertoires from single cells and seamless integration with transcriptomic data.Availability and implementationScirpy source code and documentation are available at https://github.com/icbi-lab/scirpy.

scholarly journals Growth Response and Recovery of Corynebacterium glutamicum Colonies on Single-Cell Level Upon Defined pH Stress Pulses

2021 ◽  
Vol 12 ◽  
Author(s):  
Sarah Täuber ◽  
Luisa Blöbaum ◽  
Volker F. Wendisch ◽  
Alexander Grünberger
Keyword(s):  
Single Cell ◽  
Corynebacterium Glutamicum ◽  
Intracellular Ph ◽  
Growth Response ◽  
Physiological State ◽  
Single Cell Level ◽  
Cell Level ◽  
Stress Pulse ◽  
Ph Values ◽  
Ph Stress

Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum.

scholarly journals Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0238330
Author(s):  
Tobias Gross ◽  
Csaba Jeney ◽  
Darius Halm ◽  
Günter Finkenzeller ◽  
G. Björn Stark ◽  
...  
Keyword(s):  
Single Cell ◽  
Large Scale ◽  
Single Cells ◽  
Protein Detection ◽  
High Yield ◽  
Fluorescent Properties ◽  
Cell Printing ◽  
Protein Levels ◽  
Cell Clones

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.

Characterization of the Immobilized Fructosyltranferase from Rhodotorula sp.

2010 ◽  
Vol 6 (3) ◽  
Author(s):  
Elizama Aguiar-Oliveira ◽  
Francisco Maugeri
Keyword(s):  
High Activity ◽  
Large Scale ◽  
Immobilized Enzyme ◽  
Enzyme System ◽  
Substrate Inhibition ◽  
Free Enzyme ◽  
Ph Profile ◽  
Ph Values ◽  
Sucrose Solutions

The fructosyltransferase from Rhodotorula sp. recovered from a fermented medium, by precipitation was immobilized by adsorption onto niobium ore. Considering the biocatalyst system, the activity/pH profile moved towards more alkaline values as compared to the free enzyme system, indicating that the support affected the charge distribution between the enzyme and the support. The immobilized enzyme showed high activity and good stability at pH values of 4.5 and 6.0. The biocatalyst half-lives at 48 °C and pH values of 4.5 and 6.0 were 32 and 72 days, respectively. The kinetics for the immobilized system corresponded to that of substrate inhibition. The synthesis of fructooligosaccharides from 50% sucrose solutions was carried out in batch stirred reactors and the conversion was about 58%, similar to that with the free enzyme. Based on the biocatalyst activity, stability and process yields, the system developed in this work can be considered suitable for application in large-scale bioreactors.

scholarly journals Erratum to: CO2/HCO3 − perturbations of simulated large scale gradients in a scale-down device cause fast transcriptional responses in Corynebacterium glutamicum

2014 ◽  
Vol 98 (20) ◽  
pp. 8775-8775
Author(s):  
Jens Buchholz ◽  
Michaela Graf ◽  
Andreas Freund ◽  
Tobias Busche ◽  
Jörn Kalinowski ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Large Scale ◽  
Transcriptional Responses ◽  
Scale Down

scholarly journals X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin

2021 ◽  
Vol 22 (24) ◽  
pp. 13392
Author(s):  
Camila Campos-Escamilla ◽  
Dritan Siliqi ◽  
Luis A. Gonzalez-Ramirez ◽  
Carmen Lopez-Sanchez ◽  
Jose Antonio Gavira ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Biomedical Applications ◽  
Iron Release ◽  
X Ray ◽  
X Ray Crystallography ◽  
Ph Values ◽  
X Ray Scattering ◽  
Physiological Relevance ◽  
Ph Conditions

Human serum transferrin (Tf) is a bilobed glycoprotein whose function is to transport iron through receptor-mediated endocytosis. The mechanism for iron release is pH-dependent and involves conformational changes in the protein, thus making it an attractive system for possible biomedical applications. In this contribution, two powerful X-ray techniques, namely Macromolecular X-ray Crystallography (MX) and Small Angle X-ray Scattering (SAXS), were used to study the conformational changes of iron-free (apo) and iron-loaded (holo) transferrin in crystal and solution states, respectively, at three different pH values of physiological relevance. A crystallographic model of glycosylated apo-Tf was obtained at 3.0 Å resolution, which did not resolve further despite many efforts to improve crystal quality. In the solution, apo-Tf remained mostly globular in all the pH conditions tested; however, the co-existence of closed, partially open, and open conformations was observed for holo-Tf, which showed a more elongated and flexible shape overall.

scholarly journals Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3661-3672 ◽  
Author(s):  
Guyue Cheng ◽  
Peiwei Zhao ◽  
Xiao-Feng Tang ◽  
Bing Tang
Keyword(s):  
Inclusion Bodies ◽  
Immunoblot Analysis ◽  
Gene Encoding ◽  
Ph Values ◽  
Mature Enzyme ◽  
Ph Conditions ◽  
Coat Layer ◽  
Identification And Characterization ◽  
Mature Protease

A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain. Phylogenetic analysis revealed that this enzyme belonged to the subtilisin family, but could not be grouped into any of its six known subfamilies. The mature protease CDF has an unusually high content of charged residues, which are mainly distributed on the enzyme surface. The recombinant proform of protease CDF formed inclusion bodies, but could be efficiently converted to the mature enzyme when the inclusion bodies were dissolved in alkaline buffers. The proform underwent a two-step maturation process, wherein the N-terminal part (85 residues) of the propeptide was autoprocessed intramolecularly, and the remaining 9-residue peptide was further processed intermolecularly. Protease CDF exhibited optimal proteolytic activity at 50–55 °C and pH 10.5–11.0. The enzyme was stable under high-pH conditions (pH 11.0–12.0), and NaCl could stabilize the enzyme at lower pH values. In addition, the enzyme was not dependent on calcium for either maturation or stability. By immunoblot analysis, protease CDF was found to be associated with spores, and could be extracted from the spores with 2 M KCl and alkaline buffers without damaging the coat layer, demonstrating that the protease CDF is located on the surface of the spore coat.

scholarly journals Janus Hydrogel Microbeads for Glucose Sensing with pH Calibration

Sensors ◽  
2021 ◽  
Vol 21 (14) ◽  
pp. 4829
Author(s):  
Maru Ando ◽  
Mio Tsuchiya ◽  
Shun Itai ◽  
Tomomi Murayama ◽  
Yuta Kurashina ◽  
...  
Keyword(s):  
Body Fluid ◽  
Glucose Monitoring ◽  
Glucose Sensing ◽  
Implantable Sensors ◽  
Proof Of Concept ◽  
Ph Sensors ◽  
Ph Values ◽  
Fluorescent Microbeads ◽  
Ph Conditions ◽  
Ph Calibration

We present fluorescent Janus hydrogel microbeads for continuous glucose sensing with pH calibration. The Janus hydrogel microbeads, that consist of fluorescent glucose and pH sensors, were fabricated with a UV-assisted centrifugal microfluidic device. The microbead can calibrate the pH values of its surroundings and enables accurate measurements of glucose within various pH conditions. As a proof of concept, we succeeded in obtaining the accurate value of glucose concentration in a body-fluid-like sample solution. We believe that our fluorescent microbeads, with pH calibration capability, could be applied to fully implantable sensors for continuous glucose monitoring.

scholarly journals The Tabula Sapiens: a single cell transcriptomic atlas of multiple organs from individual human donors

2021 ◽  
Author(s):  
◽  
Stephen R Quake
Keyword(s):  
Single Cell ◽  
Molecular Characterization ◽  
Rna Splicing ◽  
Detailed Comparison ◽  
Cell Types ◽  
Proliferative Potential ◽  
Tissue Specific ◽  
Multiple Organs ◽  
Cell Transcriptome

In recent years there has been tremendous progress towards deep molecular characterization of cell types using single cell transcriptome sequencing. Here we report a single cell transcriptomic atlas comprising nearly 500,000 cells from 24 different human tissues and organs. In several instances multiple organs were analyzed from the same donor. Analyzing organs from the same individual controls for genetic background, age, environment, and epigenetic effects, and enables a detailed comparison of cell types that are shared between tissues. This resource provides a rich molecular characterization of more than 400 cell types, their distribution across tissues, and detailed information about tissue specific variation in gene expression. We have used the fact that multiple tissues came from the same donor to study the clonal distribution of T cells between tissues, to understand the tissue specific mutation rate in B cells, and to analyze the cell cycle state and proliferative potential of shared cell types across tissues. Finally, we have also used this data to characterize cell type specific RNA splicing and how such splicing varies across tissues within an individual.

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