Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides

Warner, Joseph R.; Reeder, Philippa J.; Karimpour-Fard, Anis; Woodruff, Lauren B.A.; Gill, Ryan T.

doi:10.1038/nbt.1653

Cited by 265 publications

(275 citation statements)

References 50 publications

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“…However, the precise relationship between aldehydes and ROS is unclear. For example, it was recently shown that resistance of E. coli to exogenous methylglyoxal is conferred by decreased expression of sodC (84). This is a surprising result given that sodC encodes a superoxide dismutase, which breaks down ROS (85).…”

Section: Addressing Aldehyde Toxicitymentioning

confidence: 99%

Microbial Engineering for Aldehyde Synthesis

Kunjapur

Prather

2015

Appl Environ Microbiol

149

129

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Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity.

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Section: Addressing Aldehyde Toxicitymentioning

confidence: 99%

Microbial Engineering for Aldehyde Synthesis

Kunjapur

Prather

2015

Appl Environ Microbiol

149

129

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“…However, all genetic engineering methods share the same inherent problem: while they can alter a particular phenotype, they cannot simultaneously select for the most robust strain with that phenotype. Innovative screens, such as selection for colony size, have been developed that lessen the impact of this limitation, but the problem persists nevertheless (35,61). More critically, engineered alterations in one phenotype frequently come at the expense of other critical phenotypes, such as growth rate (39).…”

mentioning

confidence: 99%

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Blaby¹,

Lyons²,

Wroclawska-Hughes³

et al. 2012

Appl Environ Microbiol

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Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/ maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wildtype fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.

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“…Although our procedure requires RE manipulation, it is not limited by location, the availability of RE sites, nor addition of restriction site, but it is a rare occasion that a non-cutter can be useful. With the efficiency of targeting and the transient nature of tSM, it is conceivable that high throughput site-directed mutagenesis is possible to make a slight difference in otherwise identical plasmids, or by making random mutations in designated areas to generate pools of mutations in a defined domain [39] . Indeed, based on our experience, as long as homologous arms can form a stable complex with acceptor plasmids, single, double or even multiple mutations can be imported in one targeting event.…”

Section: Discussionmentioning

confidence: 99%

Transitory selection markers facilitate DNA mutagenesis with recombineering

Cheng

Hollenback

et al. 2015

JBEI

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Site-directed mutagenesis with DNA oligos is a powerful tool to generate subtle alterations in DNA. Taking advantage of strand replacement during lambda-red recombineering, mutations on DNA (donor) can be introduced into another DNA construct (acceptor) via targeting. We describe a novel approach that takes advantage of the specificity of homologous recombination and the convenience of positive selection to insert mutations into existing DNA constructs. With simple procedures, we generated a "transitory" selection marker that is used to carry modifications into acceptor DNA and then removed by enzyme manipulation, leaving only the desired mutation without any scar. Overall, this method of targeting mutagenesis via recombineering provides a precision manipulation of DNA and avoids the extensive PCR reaction. Not only can this method be used to make point mutations, truncations or Epitope-tags, but it can also be used to import existing mutations, generate in-frame fusion, or repair missing components.

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Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides

Cited by 265 publications

References 50 publications

Microbial Engineering for Aldehyde Synthesis

Microbial Engineering for Aldehyde Synthesis

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Transitory selection markers facilitate DNA mutagenesis with recombineering

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