Identification of Yeast Pheromone Pathway Modulators by High-Throughput Agonist Response Profiling of a Yeast Gene Knockout Strain Collection

2004 ◽  
pp. 399-409 ◽  
Author(s):  
Scott A. Chasse ◽  
Henrik G. Dohlman
Keyword(s):  
High Throughput ◽  
Gene Knockout ◽  
Yeast Gene ◽  
Knockout Strain ◽  
Strain Collection

scholarly journals Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

2019 ◽  
Author(s):  
Gabriel A. Suárez ◽  
Kyle R. Dugan ◽  
Brian A. Renda ◽  
Sean P. Leonard ◽  
Lakshmi S. Gangavarapu ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Single Gene ◽  
Golden Gate ◽  
Acinetobacter Baylyi ◽  
Gene Deletions ◽  
Multiple Gene ◽  
A Genome ◽  
Strain Collection ◽  
Acinetobacter Baylyi Adp1 ◽  
Improved Methods

ABSTRACTOne goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 19 successful multiple-gene deletions ranged in size from 21 to 183 kilobases and collectively accounted for 24.6% of its genome. Deletion success could only be partially predicted on the basis of a single-gene knockout strain collection and a new Tn-Seq experiment. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

scholarly journals Isonicotinamide extends yeast chronological lifespan through a mechanism that diminishes nucleotides

2021 ◽  
Author(s):  
Agata Kalita ◽  
Elisa Enriquez Hesles ◽  
Lindsey N Power ◽  
Dezhen Wang ◽  
Pankaj Kumar singh ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Nucleotide Metabolism ◽  
Cellular Aging ◽  
Post Inoculation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Functional Feature ◽  
Nucleotide Biosynthesis ◽  
Chronological Lifespan ◽  
Nucleotide Pools ◽  
Strain Collection

Chronological lifespan (CLS) of budding yeast, Saccharomyces cerevisiae, is a commonly utilized model for cellular aging of non-dividing cells such as neurons. CLS is strongly extended by isonicotinamide (INAM), a non-metabolized isomer of the NAD+ precursor nicotinamide (NAM), but the underlying mechanisms of lifespan extension remain uncharacterized. To identify potential biochemical INAM targets, we performed a chemical genetic screen with the yeast gene knockout (YKO) strain collection for INAM-hypersensitive mutants. Significantly enriched Gene Ontology terms that emerged included SWR1 and other transcription elongation factors, as well as metabolic pathways converging on one-carbon metabolism and contributing to nucleotide biosynthesis, together suggesting that INAM perturbs nucleotide pools. In line with this model, INAM effects on cell growth were synergistic with mycophenolic acid (MPA), which extends lifespan by reducing guanine nucleotide pools. Direct measurements of nucleotides and precursors by mass spectrometry indicated that INAM reduced nucleotides, including cAMP, at 24- and 96-hour time points post-inoculation. Taken together, we conclude that INAM extends CLS by perturbing nucleotide metabolism, which may be a common functional feature of multiple anti-aging interventions.

scholarly journals Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

2019 ◽  
Author(s):  
Srijan Jindal ◽  
Lei Yang ◽  
Philip J. Day ◽  
Douglas B. Kell
Keyword(s):  
Steady State ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Dyes ◽  
Gene Knockout ◽  
Sybr Green ◽  
Efflux Transporters ◽  
General Strategy ◽  
Individual Gene

AbstractWe used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

scholarly journals High throughput identification of genes conferring resistance or sensitivity to toxic effectors delivered by the type VI secretion system

2021 ◽  
Author(s):  
Steven J Hersch ◽  
Rehnuma T Sejuty ◽  
Kevin Manera ◽  
Tao G Dong
Keyword(s):  
High Throughput ◽  
Gene Knockout ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Vi Secretion System ◽  
Numerous Species ◽  
E Coli ◽  
Metabolic Genes ◽  
Defence Genes ◽  
Immunity Genes

The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner.

High-throughput DEXA and micro-CT screening in gene knockout mice identifies bone mass phenotypes

2013 ◽  
Author(s):  
Robert Brommage ◽  
Jeff Liu ◽  
Laura Kirkpatrick ◽  
David Powell ◽  
Peter Vogel
Keyword(s):  
Bone Mass ◽  
High Throughput ◽  
Knockout Mice ◽  
Gene Knockout ◽  
Micro Ct ◽  
Ct Screening ◽  
Gene Knockout Mice

scholarly journals Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis inHaloferax volcanii

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Ian K. Blaby ◽  
Gabriela Phillips ◽  
Crysten E. Blaby-Haas ◽  
Kevin S. Gulig ◽  
Basma El Yacoubi ◽  
...  
Keyword(s):  
High Throughput ◽  
Gene Deletion ◽  
Systems Approach ◽  
Essential Gene ◽  
Gene Knockout ◽  
Deletion Mutants ◽  
Mutant Library ◽  
Phenotypic Analysis ◽  
Genome Wide ◽  
A Genome

With the availability of a genome sequence and increasingly sophisticated genetic tools,Haloferax volcaniiis becoming a model for both Archaea and halophiles. In order forH. volcaniito reach a status equivalent toEscherichia coli, Bacillus subtilis, orSaccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22H. volcaniideletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of anH. volcaniimutant library and suggest that they should form the basis of a larger genome-wide experiment.

scholarly journals Differences in Interactions between Azole Drugs Related to Modifications in the 14-α Sterol Demethylase Gene (cyp51A) of Aspergillus fumigatus

2005 ◽  
Vol 49 (5) ◽  
pp. 2119-2121 ◽  
Author(s):  
G. Garcia-Effron ◽  
E. Mellado ◽  
A. Gomez-Lopez ◽  
L. Alcazar-Fuoli ◽  
M. Cuenca-Estrella ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Knockout ◽  
Resistance Mechanisms ◽  
Knockout Strain ◽  
Resistant Strains ◽  
Gene Knockouts

ABSTRACT The combined activity of different azole drugs was investigated. Thirty-one Aspergillus fumigatus strains were tested, including two cyp51A − and one cyp51B − gene-knockout strain and azole-susceptible and -resistant strains with different resistance mechanisms. The combination of itraconazole and voriconazole was synergistic for all strains except for those with gene knockouts.

scholarly journals Characterisation of iunH gene knockout strain from Mycobacterium tuberculosis

2017 ◽  
Vol 112 (3) ◽  
pp. 203-208 ◽  
Author(s):  
Anne Drumond Villela ◽  
Valnês da Silva Rodrigues Junior ◽  
Antônio Frederico Michel Pinto ◽  
Priscila Lamb Wink ◽  
Zilpa Adriana Sánchez-Quitian ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Gene Knockout ◽  
Knockout Strain

scholarly journals Inferences About the Distribution of Dominance Drawn From Yeast Gene Knockout Data

Genetics ◽  
2010 ◽  
Vol 187 (2) ◽  
pp. 553-566 ◽  
Author(s):  
Aneil F. Agrawal ◽  
Michael C. Whitlock
Keyword(s):  
Gene Knockout ◽  
Yeast Gene

scholarly journals A Perspective on the Future of High-Throughput RNAi Screening

2015 ◽  
Vol 20 (8) ◽  
pp. 1040-1051 ◽  
Author(s):  
Jessica Taylor ◽  
Simon Woodcock
Keyword(s):  
Functional Genomics ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Gene Knockout ◽  
Strand Break ◽  
Loss Of Function ◽  
Rnai Screening ◽  
Guide Rna ◽  
Cas A

For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA–Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery.

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