Gene editing techniques for toxin antisieve modules (TCCRAS) regulated by antitoxin switches.

Efficient gene editing technology is the core technology of basic biology and biotechnology research, and plays an increasingly important role in life science and biomedical fields.
Efficient genetic operation techniques based on bacterial and paleobacterial defense systems are the focus of research in the field of genomic editing, such as DNA methylation simulation systems (MoDMP) established using restrictive modification (RM) systems and CRISPR techniques established using regularly clustered interval short echo repeat sequences.
recently discovered that toxin-Antitoxin systems are a common defense system in primary nuclear cells by regulating cell growth and death/sleep by using coding products of toxins and antitoxin genes located on the same manipulator.
this mechanism, which specifically regulates cell growth and death, can be applied to bacterial DNA cloning, protein expression and genetic processing.
Bacillus subtilis, a pattern bacteria of Grain-positive bacteria, plays an important role in various physiological metabolism, signal regulation, and system metabolic engineering.
Although site-specific recombinant enzyme mediators, antisiefest methods and CRISPR-Cas9 gene editing techniques have been established in Bacillus aureus, the complex cell wall structure of Terrain-positive bacteria and strong cytotoxicity lead to a high false positive rate of genetic operation.
, the existing genetic methods are unable to efficiently and steadily genetically edit most Terrain-positive bacteria, such as Bacillus.
In response to the bottleneck of the lack of an efficient and stable traceless genetic operating system for Gloine-positive bacteria, the Wentingyi Research Group of the Key Laboratory of Pathogenic Microbiology and Immunology of the Institute of Microbiology of the Chinese Academy of Sciences and the Research Group of the Institute of Toxic Drugs of the Academy of Military Medical Sciences carried out a cooperative study to re-emphasise the bacterial toxin-antitoxin system. The toxin antisieve module (Toxin Counter-selectable Cassette Regulationed by an Antitoxin Switch, TCCRAS) is programmed to be regulated by an antitoxin switch and optimized for gene editing for Gram-positive bacteria such as Bacillus antelasia and Glutamate.
First of all, the toxin anti-sieve module (TCCRAS) regulated by antitoxin switch was constructed by using the strategy of starting toxin and antitoxin gene expression respectively by the composition type and induced promoter, and an efficient anti-sieve module reelBE was obtained by screening the type II TA system of five different sources, and the genetic operation carrier carrying TCCRAS system was constructed.
The system can be used to efficiently achieve the deletion, insertion, replacement, precise point mutation and insertion and deletion of large pieces of DNA (up to 194.9kb) in the dead grass bud cytobacteria, and by inserting the Pspac-crtI-crtE-crtB manipulator, the biosynthesis of lycopene in the dead grass bud cytobacteria for the first time.
In addition, the system can be used to remove, replace and remove up to 179.8kb fragments of Glutamate's chromosome genes, with antisieps and mutation efficiency of 100% and 17.9-85.9%, respectively, significantly higher than the traditional SacB system.
used this method to successfully construct the direct fermentation method of glutamate lollipolyceria engineering bacteria, for the future use of glutamate lollipop production of pyrethroide laid a foundation.
The study successfully established a new gene editing technique by constructing a toxin anti-sieve module regulated by the antitoxin switch, and realized the spotless knocking, tapping, substitution, point mutation and deletion and insertion of chromosome genes in Geland-positive bacteria.
TCCRAS method has the advantages of not introducing any markers, genetic stability, high efficiency and wide use, and can be used as an effective genetic operation tool for systematic biology and synthetic biology research.
the study was published online recently in ACS Synthetic Biology, the first author of the paper by Wu Jie, a doctoral student at the Institute of Microbiology of the Chinese Academy of Sciences, and Deng Aihua, an associate researcher, and Wen Tingyi, a researcher, and Chang Yongsheng.
the study was funded by the National High Technology Research and Development Program ("863" Program) (2014AA021203), the National Natural Science Foundation of China (31570083 and 31170103), the Science and Technology Services Network Program of the Chinese Academy of Sciences (STS Program) (KFJ-EW-STS-078) and the Key Deployment Project of the Chinese Academy of Sciences (KGZD-EW-606).
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