Initiation and Velocity of Chromosome Replication in
            <i>Escherichia coli</i>
            B/r and K-12

Bipatnath, Meeshel; Dennis, Patrick P.; Bremer, H. J.

doi:10.1128/jb.180.2.265-273.1998

Cited by 87 publications

(65 citation statements)

References 29 publications

(28 reference statements)

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“…We observed a linear decrease in expression for both sfGFP (pearson r sfGFP,arm1 = −0.91, pearson r sfGFP,arm2 = −0.65, p sfGFP < 0.05, permutation test) and mKate2 (pearson r mKate,arm1 = −0.85, pearson r mKate,arm2 = 0.51, p mKate < 0.05, permutation test) reporters with respect to distance from the chromosomal origin (Figure 3B). This result was consistent with expected variations in local chromosomal copy number due to bi-directional replication dynamics during growth (45, 46). Interestingly, we observed a much stronger correlation of expression to distance from replication origin for chromosome Arm 1, though mKate2 expression at Safe Site 8 was a low outlier.…”

Section: Resultssupporting

confidence: 91%

A versatile platform strain for high-fidelity multiplex genome editing

Egbert

Rishi

Adler

et al. 2018

Preprint

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21Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the 22 engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have 23 enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed 24 mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date 25 been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of 26 inducible transcriptional regulators in these strains also prevents concurrent control of genome 27 engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, 28BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity 29 and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination 30 efficiency, and (3) four independent inducible regulators to control engineered functions. These 31 modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold 32 increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To 33 facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci,34 termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is 35 a platform to develop and optimize engineered cellular functions and can serve as a model to implement 36 comparable recombination and regulatory systems in other bacteria. 37 INTRODUCTION 38The design-build-test (DBT) cycle is a common paradigm used in engineering disciplines. Within the 39 context of synthetic biology it is employed to engineer user-defined cellular functions for applications 40 such as metabolic engineering, biosensing, and therapeutics (1, 2). The rapid prototyping of engineered 41 functions has been facilitated by advances in in vitro DNA assembly, and plasmids have traditionally 42 been used to implement designs in vivo given their ease-of-assembly and portability. However, for 43 deployment in contexts beyond the laboratory such as large-scale industrial bioprocesses or among 44 complex microbial communities, plasmid-based circuits suffer from multiple limitations: high intercellular 45 variation in gene expression, genetic instability from random partitioning of plasmids during cell division, 46 and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or 47 is economically infeasible (3, 4). These shortcomings can be ameliorated once a design is transferred 48 from a plasmid to the host genome, which offers improved genetic stability and lower expression 49 variation (5) along with reduced metabolic load (6). However, behaviors optimized for plasmid contexts 50 often do not map predictably to the genome. As such, building and testing designs directly on the 51 genome can reduce the DBT cycle time and facilitate engineering cellular programs for complex 52 e...

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Section: Resultssupporting

confidence: 91%

A versatile platform strain for high-fidelity multiplex genome editing

Egbert

Rishi

Adler

et al. 2018

Preprint

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“…Total DNA quantification was performed according to Burton (1956), using calf thymus DNA as standard. The cells were previously collected on glass fibre filters, as described by Bipatnath et al (1998). All determinations were performed at least in duplicate.…”

Section: Kinetics Of Sac Productionmentioning

confidence: 99%

Surface-active compounds and their role in the access to hydrocarbons in Gordonia strains

Franzetti

Bestetti

Caredda

et al. 2008

FEMS Microbiology Ecology

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Three new bacterial strains (M22, BS25 and BS29) belonging to the Gordonia genus were isolated from a site chronically contaminated by diesel. Those Gordonia strains were able to grow using a wide range of straight and branched aliphatic hydrocarbons as carbon and energy sources and to produce at least two classes of surface-active compounds. Emulsifying agents were released in the culture medium when bacteria grew both on hydrocarbons and water-soluble substrates. Cell-bound biosurfactants, which reduce the surface tension, were produced on hydrocarbons; however, their production was significantly lower on water soluble substrates. The relationship of growth phase, surface-active compound production and cell-surface properties was analyzed in kinetic experiments on hydrocarbons. Gordonia sp. BS29 synthesized, and released extracellularly, bioemulsans during the exponential phase with n-hexadecane as carbon and energy source. The production of biosurfactants started in the exponential phase and their concentration increased during the following linear growth. Furthermore, the adhesion of bacterial cells to hydrocarbons decreased during growth. Our results led us to hypothesize a change in the mode by which Gordonia cells access the substrate during growth on hydrocarbons.

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“…Generation times that are shorter than the C+D periods are accommodated by overlapping replication cycles such that each cell receives 2, 4 or 8 origins at birth (Fig 1). All origins in a cell fire synchronously (Skarstad et al, 1986) at a fixed cell size per origin (initiation mass) that is independent of the growth rate (Bipatnath et al, 1998;Donachie, 1968).…”

Section: The Cell Cycle and Chromosome Replicationmentioning

confidence: 99%

Copy‐number control of the Escherichia coli chromosome: a plasmidologist's view

Nordström

Dasgupta

2006

EMBO Reports

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The homeostatic system that sets the copy number, and corrects over‐replication and under‐replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy‐number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative‐feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number.

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Initiation and Velocity of Chromosome Replication in Escherichia coli B/r and K-12

Cited by 87 publications

References 29 publications

A versatile platform strain for high-fidelity multiplex genome editing

A versatile platform strain for high-fidelity multiplex genome editing

Surface-active compounds and their role in the access to hydrocarbons in Gordonia strains

Copy‐number control of the Escherichia coli chromosome: a plasmidologist's view

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