Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius
Joint Authors
Source
Issue
Vol. 2017, Issue 2017 (31 Dec. 2017), pp.1-12, 12 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2017-10-31
Country of Publication
Egypt
No. of Pages
12
Main Subjects
Abstract EN
Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools.
However, plasmid construction typically requires several steps.
Alternatively, PCR tailing for high-throughput gene disruption was also developed in S.
acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system.
In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions.
This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER).
In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion.
As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S.
acidocaldarius.
In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established.
The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S.
acidocaldarius.
American Psychological Association (APA)
Suzuki, Shoji& Kurosawa, Norio. 2017. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. Archaea،Vol. 2017, no. 2017, pp.1-12.
https://search.emarefa.net/detail/BIM-1125470
Modern Language Association (MLA)
Suzuki, Shoji& Kurosawa, Norio. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. Archaea No. 2017 (2017), pp.1-12.
https://search.emarefa.net/detail/BIM-1125470
American Medical Association (AMA)
Suzuki, Shoji& Kurosawa, Norio. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. Archaea. 2017. Vol. 2017, no. 2017, pp.1-12.
https://search.emarefa.net/detail/BIM-1125470
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-1125470