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 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 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.

High-throughput, functional screening of the anti-HIV-1 humoral response by an enzymatic nanosensor

2006 ◽  
Vol 43 (13) ◽  
pp. 2119-2123 ◽  
Author(s):  
Rosa María Ferraz ◽  
Anna Arís ◽  
Miguel Angel Martínez ◽  
Antonio Villaverde
Keyword(s):  
High Throughput ◽  
Humoral Response ◽  
Functional Screening ◽  
Anti Hiv ◽  
Hiv 1

scholarly journals High-throughput Screening and Sensitized Bacteria Identify an M. tuberculosis Dihydrofolate Reductase Inhibitor with Whole Cell Activity

PLoS ONE ◽  
2012 ◽  
Vol 7 (6) ◽  
pp. e39961 ◽  
Author(s):  
Anuradha Kumar ◽  
Meng Zhang ◽  
Linyun Zhu ◽  
Reiling P. Liao ◽  
Charles Mutai ◽  
...  
Keyword(s):  
High Throughput ◽  
Dihydrofolate Reductase ◽  
High Throughput Screening ◽  
Reductase Inhibitor ◽  
Cell Activity ◽  
Whole Cell

scholarly journals Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels

ACS Omega ◽  
2018 ◽  
Vol 3 (11) ◽  
pp. 14814-14823 ◽  
Author(s):  
Katrin M. Krebs ◽  
Eva M. Pfeil ◽  
Katharina Simon ◽  
Manuel Grundmann ◽  
Felix Häberlein ◽  
...  
Keyword(s):  
Potassium Channels ◽  
G Protein ◽  
High Throughput ◽  
Label Free ◽  
Whole Cell ◽  
Inwardly Rectifying Potassium Channels ◽  
Inwardly Rectifying

scholarly journals A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format

PLoS ONE ◽  
2015 ◽  
Vol 10 (10) ◽  
pp. e0140691 ◽  
Author(s):  
Xiaodong Xiao ◽  
Yan Chen ◽  
Sheila Mugabe ◽  
Changshou Gao ◽  
Christine Tkaczyk ◽  
...  
Keyword(s):  
High Throughput ◽  
Functional Screening ◽  
Expression Platform ◽  
Phage Libraries ◽  
Dual Expression

High-Throughput Functional Screening of GenesIn Planta

2008 ◽  
pp. 113-136 ◽  
Author(s):  
Thomas Berberich ◽  
Yoshihiro Takahashi ◽  
Hiromasa Saitoh ◽  
Ryohei Terauchi
Keyword(s):  
High Throughput ◽  
Functional Screening
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