Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

Akhverdyan, V. Z.; Gak, Evgueni R.; Tokmakova, Irina L.; Stoynova, Nataliya V.; Yomantas, Yurgis A. V.; Mashko, Sergey V.

doi:10.1007/s00253-011-3416-y

Cited by 32 publications

(29 citation statements)

References 130 publications

(194 reference statements)

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“…Since the mid-1980s, mini-Mu derivatives have been used extensively in vivo for insertional mutagenesis, for gene fusion and mapping, as well as for gene cloning and DNA sequencing strategies, including metabolic engineering (see (9, 133)). More recently, the Mu enhancer element has been either supplied in trans or excised from mini-Mu vectors to control their transposition efficiency (reviewed in (133).…”

Section: Mu As a Tool For Gene Manipulationmentioning

confidence: 99%

“…More recently, the Mu enhancer element has been either supplied in trans or excised from mini-Mu vectors to control their transposition efficiency (reviewed in (133). Electroporation of in vitro assembled and cleaved Mu R1-R2 transpososomes has been used successfully for Mu integration in a variety of bacterial species, both Gram-positive and Gram-negative (134, 135), as well as in yeast and in mammalian genomes (136).…”

Section: Mu As a Tool For Gene Manipulationmentioning

confidence: 99%

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Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

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Section: Mu As a Tool For Gene Manipulationmentioning

confidence: 99%

Section: Mu As a Tool For Gene Manipulationmentioning

confidence: 99%

Transposable Phage Mu

Harshey

2014

Microbiol Spectr

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“…Over 20 years, genetic and molecular characterization of Mu revealed its unique properties, which among other developments lead to the engineering of a number of very widely used "genetic tools" for E. coli and other Enterobacteria (Akhverdyan et al, 2011;Ferrieres et al, 2010 for a recent development; Van Gijsegem et al, 1987). In the middle 80's, the phage became the paradigm for the biochemical in vitro deciphering of the molecular mechanism of transposition catalyzed by the "DDE recombinases" (reviewed in Harshey, 2012).…”

Section: Introductionmentioning

confidence: 97%

A structured annotation frame for the transposable phages: A new proposed family “Saltoviridae” within the Caudovirales

et al. 2015

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Enterobacteriophage Mu is the best studied and paradigm member of the transposable phages. Mu-encoded proteins have been annotated in detail in UniProtKB and linked to a controlled vocabulary describing the various steps involved in the phage lytic and lysogenic cycles. Transposable phages are ubiquitous temperate bacterial viruses with a dsDNA linear genome. Twenty-six of them, that infect α, β and γ-proteobacteria, have been sequenced. Their conserved properties are described. Based on these characteristics, we propose a reorganization of the Caudovirales, to allow for the inclusion of a "Saltoviridae" family and two newly proposed subfamilies, the "Myosaltovirinae" and "Siphosaltovirinae". The latter could temporarily be included in the existing Myoviridae and Siphoviridae families.

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“…Chromosomal integration in E. coli may be achieved through use of transposons (either random or site-specific; [18,19]) or recombination mediated by phages/phage-derived elements [20-25]. Phages can be used to integrate large DNA fragments.…”

Section: Introductionmentioning

confidence: 99%

Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

et al. 2013

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BackgroundMetabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic.ResultsA series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional.ConclusionsThe KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.

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Application of the bacteriophage Mu-driven system for the integration/amplification of target genes in the chromosomes of engineered Gram-negative bacteria—mini review

Cited by 32 publications

References 130 publications

Transposable Phage Mu

Transposable Phage Mu

A structured annotation frame for the transposable phages: A new proposed family “Saltoviridae” within the Caudovirales

Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

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