Designed Gene Amplification on the Bacillus subtilis Chromosome

Mori, Masaki; Hashiguchi, Ken-ichi; Yoda, Koji; Yamasaki, Makari

doi:10.1099/00221287-134-1-85

Cited by 5 publications

(4 citation statements)

References 30 publications

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“…Most of the cases analyzed adhere closely to the basic characteristics of the canonical model. Such examples include Bacillus subtilis (34,43,67,102,108,111,113), Deinococcus radiodurans (62), Haemophilus influenzae (14,45,51,100,101), Klebsiella aerogenes (69), Pseudomonas aeruginosa (17), Rhizobium etli (27,89,90), Streptococcus faecalis (109,110), and Vibrio cholerae (31,65). The central part of Figure 1 (boxed) represents the basic features of the canonical model, as described below.…”

Section: The Canonical Modelmentioning

confidence: 99%

Gene Amplification and Genomic Plasticity in Prokaryotes

Romero¹,

Palacios²

1997

Annu. Rev. Genet.

210

150

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Gene amplification is a common feature of the genome of prokaryotic organisms. In this review, we analyze different instances of gene amplification in a variety of prokaryotes, including their mechanisms of generation and biological role. Growing evidence supports the concept that gene amplification be considered not as a mutation but rather as a dynamic genomic state related to the adaptation of bacterial populations to changing environmental conditions or biological interactions. In this context, the potentially amplifiable DNA regions impose a defined dynamic structure on the genome. If such structure has indeed been selected during evolution, it is a particularly challenging hypothesis.

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Section: The Canonical Modelmentioning

confidence: 99%

Gene Amplification and Genomic Plasticity in Prokaryotes

Romero¹,

Palacios²

1997

Annu. Rev. Genet.

210

150

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“…2) Based on the junction structure, we had constructed a new junction structure by in vitro ligation of two DNA fragments and succeeded in inducing a 'designed' gene amplification. 3) In this report we describe the induction of another type of gene amplification with a 8.7-kb repeating unit by using the 6.4-kb EcoRI fragment, and the successful amplification of the cat gene on B. subtilis chromosome by this method.…”

Section: Induction Of Two Types Of Gene Amplification By a Cloned Dnamentioning

confidence: 97%

Induction of Two Types of Gene Amplification by a Cloned DNA Fragment and Amplification of a Foreign Gene on theBacillus subtilisChromosome by Using the Fragment

Mori

Koyama

Shiozuka

et al. 1993

Bioscience, Biotechnology, and Biochemistry

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In our previous work we cloned on λ Charon4A phage vector a 6.4-kb EcoRI fragment, which had the transforming activity of inducing gene amplification with a 16-kb repeating unit on the Bacillus subtilis chromosome by competence transformation; those transformants were selected as tunicamycin resistant (Tm(r)) transformants. We found that this DNA fragment can induce another type of gene amplification with a 8.7-kb repeating unit by transformation when we use an amyE07 mutant as a recipient cell and select both Tm(r) and amyE(+) transformants. In the latter (8.7-kb type) gene amplification, the copy number was increased up to 20 in contrast to about 10 copies in the former 16-kb type. We replaced a part of the 6.4kb EcoRI fragment with a cat gene as a model of a foreign gene, and succeeded in the amplification of the cat gene on the chromosome by the latter type of transformation.

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“…A drawback of these vectors, based mostly on rolling circle replication is their structural and segregation instability (Fleming and Patching 1994; Shoham and Demain 1991) and the need of antibiotics for selection of plasmid maintenance. The integration of the target gene into the chromosome ensures high stability but due to the single copy of the gene gives less yield of product (Janniere et al 1985; Mori et al 1988; Motejadded and Altenbuchner 2007). This limitation can be overcome by integration of tandemly amplified target genes or by integration of multiple copies of the target gene at various positions of the chromosome (Huang et al 2017; Kiel et al 1995; Petit et al 1990, 1992; Slugeňová et al 1993; van der Laan et al 1991; Wang et al 2004; Yomantas et al 2011; Young 1984).…”

Section: Introductionmentioning

confidence: 99%

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system

Watzlawick

Altenbuchner

2019

AMB Expr

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The ganA gene from Bacillus subtilis encoding a β-galactosidase for degradation of the galactomannan was integrated in different loci of the B. subtilis chromosome employing the CRISPR/Cas9 system. Hereby a total of five copies of ganA cassettes in which the ganA gene was fused with the glucitol-promoter were inserted in the recipient chromosome wherein hypothetical, sporulation and protease genes were deleted. The strain with five copies of ganA expression cassette showed a β-galactosidase activity similar to the one with the same gene on a pUB110 derived multi-copy plasmid and under the same regulatory control of the glucitol promoter and GutR activator. The production of β-galactosidase in the strain with the multi-copy plasmid decreased rapidly when growth was performed under induced conditions and without antibiotic selection. In contrast, the strain with the five copies of ganA in the chromosome produced β-galactosidase for at least 40 generations. This demonstrates that the CRISPR/Cas9 system is a valuable and easy tool for constructing stable producer strains. The bigger efforts that are needed for the multiple target gene integration into the chromosome compared to cloning in expression vectors were justified by the higher stability of the target genes and the lack of antibiotic resistance genes.

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Designed Gene Amplification on the Bacillus subtilis Chromosome

Cited by 5 publications

References 30 publications

Gene Amplification and Genomic Plasticity in Prokaryotes

Gene Amplification and Genomic Plasticity in Prokaryotes

Induction of Two Types of Gene Amplification by a Cloned DNA Fragment and Amplification of a Foreign Gene on theBacillus subtilisChromosome by Using the Fragment

Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system

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