<i>E. coli</i> Genome Manipulation by P1 Transduction

Thomason, Lynn C.; Costantino, Nina; Court, Donald L.

doi:10.1002/0471142727.mb0117s79

Cited by 561 publications

(481 citation statements)

References 7 publications

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“…Although the maximum size tested in this study was 59 kb, we expect this methodology to have the capacity to incorporate even larger pieces with minimal impact on efficiency. Phage delivery can mediate the insertion of pieces up to 90 kb in size, an upper bound imposed by the amount of DNA packaged into the head of a bacteriophage particle 39 . Plasmid delivery has even less stringent limitations as it only requires stable maintenance of the genetic fragment on a plasmid or BAC, the latter of which has been routinely used for genetic fragments as large as 300 kb in size 40 .…”

Section: Discussionmentioning

confidence: 99%

“…BAL 1075 was transformed with pALG3.4 and used for the preparation of lysates from the P1vir bacteriophage 39,42 . This lysate was subsequently used to infect an overnight culture of BAL 1075 ldhA::loxP-cat-lox5171 pJW168 grown at 30°C in LB medium with ampicillin and 1 mM IPTG.…”

Section: Methodsmentioning

confidence: 99%

“…Transfer of a second integrated copy of ALG3.4 (into BAL 1450 to generate BAL 1493 or into BAL 1453 to generate BAL 1452) was mediated by P1vir phage transduction 39,42 . Proper integration was verified by colony PCR as described above.…”

Section: Methodsmentioning

confidence: 99%

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Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Santos

Regitsky²,

Yoshikuni³

2013

Nat Commun

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Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable instalment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a B40% higher titre than its plasmidbased counterpart and enables substantial improvements in titre (B330%) and productivity (B1,200%) after 50 generations.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Santos

Regitsky²,

Yoshikuni³

2013

Nat Commun

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“…Plasmids were constructed in E. coli DH5a or BL21DE3 and then electroporated into P. gingivalis strain W83 using previously described methods (AlbertiSegui et al, 2010;Davey & Duncan, 2006). P1 transduction, using hup alleles provided by Oberto et al (2009), was performed as described by Thomason et al (2007) to isolate the various hup mutants. E. coli strains were grown in Luria-Bertani broth (LB) and maintained on LB containing 1.5 % w/v agar (LB agar) at 37 uC.…”

Section: Methodsmentioning

confidence: 99%

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

et al. 2013

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HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoidassociated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUa (PG1258) and the HUb (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUb using comparative expression profiling of the PG0121 (HUb) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUb has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake. INTRODUCTIONPorphyromonas gingivalis is a Gram-negative obligate anaerobe belonging to the family Bacteroidaceae that persists as a natural member of the human oral microbiota. A shift in the microbial community leading to outgrowth of this anaerobe is directly linked to periodontitis, a chronic inflammatory disease that leads to destruction of the tissues supporting the gums and ultimately, exfoliation of the teeth (Choil et al., 1990;Dzink et al., 1988;Grossi et al., 1994;Lamont & Jenkinson, 2000;Moore et al., 1991). This commensal can colonize, invade and multiply within gingival epithelial cells, as well as penetrate into deeper epithelial cell layers, potentially releasing the whole organism and/or virulence factors into the bloodstream (reviewed by Yilmaz, 2008). In addition to its ability to cause disease in the oral cavity, there are data indicating a role in systemic disease, including its ability to invade vascular endothelial cells (Dorn et al., 2000(Dorn et al., , 2002Jandik et al., 2008) and to cause aggregation of platelets (Pham et al., 2002). For many pathogenic bacteria, surface polysaccharides play a key role in immune modulation et al., 1996). HU typically acts as an accessory protein in virtually all types of nucleoprotein-mediated processes (reviewed by...

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“…Amino-acid-overproducing mutants were identified using CASOP-GS (Supplementary Methods). E. coli BW25113 (Baba et al, 2006) was used as wild type (WT), into which deletion alleles from existing strains (Baba et al, 2006) or the arabinose utilisation locus (Ara þ ) from E. coli strain REL 607 (Lenski et al, 1991) were introduced by P1 transduction (Thomason et al, 2007). The ability to utilise arabinose was used as a phenotypic marker to identify strains in multi-strain competition experiments.…”

Section: Strain Constructionmentioning

confidence: 99%

Fitness and stability of obligate cross-feeding interactions that emerge upon gene loss in bacteria

et al. 2013

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Cross-feeding interactions, in which bacterial cells exchange costly metabolites to the benefit of both interacting partners, are very common in the microbial world. However, it generally remains unclear what maintains this type of interaction in the presence of non-cooperating types. We investigate this problem using synthetic cross-feeding interactions: by simply deleting two metabolic genes from the genome of Escherichia coli, we generated genotypes that require amino acids to grow and release other amino acids into the environment. Surprisingly, in a vast majority of cases, cocultures of two cross-feeding strains showed an increased Darwinian fitness (that is, rate of growth) relative to prototrophic wild type cells-even in direct competition. This unexpected growth advantage was due to a division of metabolic labour: the fitness cost of overproducing amino acids was less than the benefit of not having to produce others when they were provided by their partner. Moreover, frequency-dependent selection maintained cross-feeding consortia and limited exploitation by non-cooperating competitors. Together, our synthetic study approach reveals ecological principles that can help explain the widespread occurrence of obligate metabolic cross-feeding interactions in nature.

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E. coli Genome Manipulation by P1 Transduction

Cited by 561 publications

References 7 publications

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

Fitness and stability of obligate cross-feeding interactions that emerge upon gene loss in bacteria

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