Measuring growth rate in high-throughput growth phenotyping

Blomberg, Anders

doi:10.1016/j.copbio.2010.10.013

Cited by 60 publications

(54 citation statements)

References 61 publications

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“…The first problem is the neighbor effect. To date arrayed-colonies have been used for high-throughput growth quantification because experiment throughput is dramatically increased by manipulating high-density arrayed colonies (1536 colonies/plate) using robotic technology [6]. Since this method can provide cost-effective growth measurement, high-density arrays have been widely used in phenotype screening studies of genome-wide mutant library [1-5].…”

Section: Introductionmentioning

confidence: 99%

Colony-live — a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli

et al. 2014

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BackgroundPrecise quantitative growth measurements and detection of small growth changes in high-throughput manner is essential for fundamental studies of bacterial cell. However, an inherent tradeoff for measurement quality in high-throughput methods sacrifices some measurement quality. A key challenge has been how to enhance measurement quality without sacrificing throughput.ResultsWe developed a new high-throughput measurement system, termed Colony-live. Here we show that Colony-live provides accurate measurement of three growth values (lag time of growth (LTG), maximum growth rate (MGR), and saturation point growth (SPG)) by visualizing colony growth over time. By using a new normalization method for colony growth, Colony-live gives more precise and accurate growth values than the conventional method. We demonstrated the utility of Colony-live by measuring growth values for the entire Keio collection of Escherichia coli single-gene knockout mutants. By using Colony-live, we were able to identify subtle growth defects of single-gene knockout mutants that were undetectable by the conventional method quantified by fixed time-point camera imaging. Further, Colony-live can reveal genes that influence the length of the lag-phase and the saturation point of growth.ConclusionsMeasurement quality is critical to achieving the resolution required to identify unique phenotypes among a diverse range of phenotypes. Sharing high-quality genome-wide datasets should benefit many researchers who are interested in specific gene functions or the architecture of cellular systems. Our Colony-live system provides a new powerful tool to accelerate accumulation of knowledge of microbial growth phenotypes.

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Section: Introductionmentioning

confidence: 99%

Colony-live — a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli

et al. 2014

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“…Such studies should seek to address how common such pleiotropic effects are and whether their qualitative and quantitative effects on fitness can contribute to the maintenance of low wild-type mutation rates in natural populations. High-throughput growth rate screens (Blomberg, 2011;Hall et al, 2014) of mutant libraries could be conducted as a first approximation of fitness effects of mutation rate modifiers in model organisms such as yeast and E. coli; such growth rate measurements, however, may not capture all aspects of competitive fitness and may differ significantly in different propagation conditions (for example, MutS may be directly beneficial to bacteria in oxidizing environments: Torres-Barceló et al, 2013). Any comprehensive investigation of potential direct mutator fitness effects in asexual populations, moreover, will be complicated by the difficulty of separating direct fitness effects from indirect selective effects due to associated mutations.…”

Section: Future Directionsmentioning

confidence: 99%

Experimental evolution and the dynamics of genomic mutation rate modifiers

Raynes

Sniegowski

2014

Heredity

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Because genes that affect mutation rates are themselves subject to mutation, mutation rates can be influenced by natural selection and other evolutionary forces. The population genetics of mutation rate modifier alleles has been a subject of theoretical interest for many decades. Here, we review experimental contributions to our understanding of mutation rate modifier dynamics. Numerous evolution experiments have shown that mutator alleles (modifiers that elevate the genomic mutation rate) can readily rise to high frequencies via genetic hitchhiking in non-recombining microbial populations. Whereas these results certainly provide an explanatory framework for observations of sporadically high mutation rates in pathogenic microbes and in cancer lineages, it is nonetheless true that most natural populations have very low mutation rates. This raises the interesting question of how mutator hitchhiking is suppressed or its phenotypic effect reversed in natural populations. Very little experimental work has addressed this question; with this in mind, we identify some promising areas for future experimental investigation.

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“…Bacterial growth is measured by optical density (in liquid medium) or colony size (on solid medium). Hundreds of microtiter plates, robotic equipment, and specific standardization methods are required to minimize the systematic variation and batch effects across plates (22). Using an Illumina next-generationsequencing platform (23), a method to profile a library of loss-of-function Saccharomyces cerevisiae strains in response to small molecules was developed (25).…”

mentioning

confidence: 99%

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia

Gislason

Choy

Bloodworth

et al. 2017

Antimicrob Agents Chemother

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Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

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Measuring growth rate in high-throughput growth phenotyping

Cited by 60 publications

References 61 publications

Colony-live — a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli

Colony-live — a high-throughput method for measuring microbial colony growth kinetics— reveals diverse growth effects of gene knockouts in Escherichia coli

Experimental evolution and the dynamics of genomic mutation rate modifiers

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia

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