Construction and manipulation of giant DNA by a genome vector

Mitsuhiro Itaya, Kenji Tsuge

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.

Original languageEnglish
Pages (from-to)427-447
Number of pages21
JournalMethods in Enzymology
Volume498
DOIs
Publication statusPublished - 2011

Fingerprint

Genes
Genome
DNA
Bacillus subtilis
Organism Cloning
Cloning
Bacilli
Eubacterium
Synechocystis
Genetic Vectors
Gene Regulatory Networks
Homologous Recombination
Biotechnology
Bacillus
DNA sequences
Metabolites
RNA
Escherichia coli
Bacteria
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Construction and manipulation of giant DNA by a genome vector. / Itaya, Mitsuhiro; Tsuge, Kenji.

In: Methods in Enzymology, Vol. 498, 2011, p. 427-447.

Research output: Contribution to journalArticle

Itaya, Mitsuhiro ; Tsuge, Kenji. / Construction and manipulation of giant DNA by a genome vector. In: Methods in Enzymology. 2011 ; Vol. 498. pp. 427-447.
@article{549b63ceae654b30a3bc2a0f917ab056,
title = "Construction and manipulation of giant DNA by a genome vector",
abstract = "Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.",
author = "Mitsuhiro Itaya and Kenji Tsuge",
year = "2011",
doi = "10.1016/B978-0-12-385120-8.00019-X",
language = "English",
volume = "498",
pages = "427--447",
journal = "Methods in Enzymology",
issn = "0076-6879",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Construction and manipulation of giant DNA by a genome vector

AU - Itaya, Mitsuhiro

AU - Tsuge, Kenji

PY - 2011

Y1 - 2011

N2 - Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.

AB - Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.

UR - http://www.scopus.com/inward/record.url?scp=79957476633&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79957476633&partnerID=8YFLogxK

U2 - 10.1016/B978-0-12-385120-8.00019-X

DO - 10.1016/B978-0-12-385120-8.00019-X

M3 - Article

C2 - 21601689

AN - SCOPUS:79957476633

VL - 498

SP - 427

EP - 447

JO - Methods in Enzymology

JF - Methods in Enzymology

SN - 0076-6879

ER -