scholarly journals CRAGE-mediated insertion of fluorescent chromosomal markers for accurate and scalable measurement of co-culture dynamics in Escherichia coli

2020 ◽  
Author(s):  
Avery J C Noonan ◽  
Yilin Qiu ◽  
Joe C H Ho ◽  
Jewel Ocampo ◽  
K A Vreugdenhil ◽  
...  
Keyword(s):  
Population Dynamics ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Functional Screening ◽  
Whole Cell ◽  
Cell Abundance ◽  
Gfp Fluorescence ◽  
Biosensor Strain ◽  
Functional Screens ◽  
The Relationship

Abstract Monitoring population dynamics in co-culture is necessary in engineering microbial consortia involved in distributed metabolic processes or biosensing applications. However, it remains difficult to measure strain-specific growth dynamics high-throughput formats. This is especially vexing in plate-based functional screens leveraging whole-cell biosensors to detect specific metabolic signals. Here we develop an experimental high-throughput co-culture system to measure and model the relationship between fluorescence and cell abundance, combining chassis-independent recombinase-assisted genome engineering (CRAGE) and whole-cell biosensing with a PemrR-green fluorescent protein (GFP) monoaromatic reporter used in plate-based functional screening. CRAGE was used to construct E. coli EPI300 strains constitutively expressing red fluorescent protein (RFP) and the relationship between RFP expression and optical density (OD600) was determined throughout the EPI300 growth cycle. A linear equation describing the increase of normalized RFP fluorescence during deceleration phase was derived and used to predict biosensor strain dynamics in co-culture. Measured and predicted values were compared using flow cytometric detection methods. Induction of the biosensor lead to increased GFP fluorescence normalized to biosensor cell abundance, as expected, but a significant decrease in relative abundance of the biosensor strain in co-culture and a decrease in bulk GFP fluorescence. Taken together, these results highlight sensitivity of population dynamics to variations in metabolic activity in co-culture and the potential effect of these dynamics on the performance of functional screens in plate-based formats. The engineered strains and model used to evaluate these dynamics provide a framework for optimizing growth of synthetic co-cultures used in screening, testing and pathway engineering applications

scholarly journals Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries

2016 ◽  
Vol 7 ◽  
Author(s):  
Rubén Zapata-Pérez ◽  
Antonio G. García-Saura ◽  
Mohamed Jebbar ◽  
Peter N. Golyshin ◽  
Álvaro Sánchez-Ferrer
Keyword(s):  
High Throughput ◽  
Functional Screening ◽  
Whole Cell

scholarly journals Functional Screening Assays with Neurons Generated from Pluripotent Stem Cell–Derived Neural Stem Cells

2013 ◽  
Vol 19 (1) ◽  
pp. 32-43 ◽  
Author(s):  
Anastasia Efthymiou ◽  
Atossa Shaltouki ◽  
Joseph P. Steiner ◽  
Balendu Jha ◽  
Sabrina M. Heman-Ackah ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Patient Specific ◽  
Neural Cells ◽  
Functional Screening ◽  
Neurite Length ◽  
Screening Assays ◽  
Green Fluorescent ◽  
High Throughput Screens

Rapid and effective drug discovery for neurodegenerative disease is currently impeded by an inability to source primary neural cells for high-throughput and phenotypic screens. This limitation can be addressed through the use of pluripotent stem cells (PSCs), which can be derived from patient-specific samples and differentiated to neural cells for use in identifying novel compounds for the treatment of neurodegenerative diseases. We have developed an efficient protocol to culture pure populations of neurons, as confirmed by gene expression analysis, in the 96-well format necessary for screens. These differentiated neurons were subjected to viability assays to illustrate their potential in future high-throughput screens. We have also shown that organelles such as nuclei and mitochondria could be live-labeled and visualized through fluorescence, suggesting that we should be able to monitor subcellular phenotypic changes. Neurons derived from a green fluorescent protein–expressing reporter line of PSCs were live-imaged to assess markers of neuronal maturation such as neurite length and co-cultured with astrocytes to demonstrate further maturation. These studies confirm that PSC-derived neurons can be used effectively in viability and functional assays and pave the way for high-throughput screens on neurons derived from patients with neurodegenerative disorders.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Functional Genomic Identification of Cadmium Resistance Genes from a High GC Clone Library by Coupling the Sanger and PacBio Sequencing Strategies

Genes ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 7
Author(s):  
Jinghao Chen ◽  
Chao Xing ◽  
Xin Zheng ◽  
Xiaofang Li
Keyword(s):  
High Throughput ◽  
Resistance Genes ◽  
Sanger Sequencing ◽  
Genomic Library ◽  
Full Length ◽  
Host Cells ◽  
Full Genome Sequence ◽  
Functional Screening ◽  
Functional Genomic ◽  
Pacbio Sequencing

Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to Escherichia coli subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.

Alix (AIP1) is a vasopressin receptor (V2R)-interacting protein that increases lysosomal degradation of the V2R

2007 ◽  
Vol 292 (5) ◽  
pp. F1303-F1313 ◽  
Author(s):  
Xianhua Yi ◽  
Richard Bouley ◽  
Herbert Y. Lin ◽  
Shaliha Bechoua ◽  
Tian-xiao Sun ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Human Kidney ◽  
Lysosomal Degradation ◽  
Interacting Protein ◽  
Parathyroid Hormone Receptor ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Regulatory Event

The vasopressin type 2 receptor (V2R) is a G protein-coupled receptor that plays a central role in renal water reabsorption. Termination of ligand (vasopressin) stimulation is an important physiological regulatory event, but few proteins that interact with the V2R during downregulation after vasopressin (VP) binding have been identified. Using yeast two-hybrid screening of a human kidney cDNA library, we show that a 100-kDa protein called ALG-2-interacting protein X (Alix) interacts with the last 29 amino acids of the V2R COOH terminus. This was confirmed by pull-down assays using a GST-V2R-COOH-tail fusion protein. Alix was immunolocalized in principal cells of the kidney, which also express the V2R. The function of the Alix-V2R interaction was studied by transfecting Alix into LLC-PK1 epithelial cells expressing V2R-green fluorescent protein (GFP). Under basal conditions, V2R-GFP localized mainly at the plasma membrane. On VP treatment, V2R-GFP was internalized into perinuclear vesicles in the nontransfected cells. In contrast, V2R-GFP fluorescence was virtually undetectable 2 h after exposure to VP in cells that coexpressed Alix. Western blotting using an anti-GFP antibody showed marked degradation of the V2R after 2 h in the presence of VP and Alix, a time point at which little or no degradation was detected in the absence of Alix. In contrast, little or no degradation of the parathyroid hormone receptor was detectable in the presence or absence of Alix and/or the PTH ligand. The VP-induced disappearance of V2R-GFP was abolished by chloroquine, a lysosomal degradation inhibitor, but not by MG132, a proteosome inhibitor. These data suggest that Alix increases the rate of lysosomal degradation of V2R and may play an important regulatory role in the VP response by modulating V2R downregulation.

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