Construction of seamless genome editing system for fast-growing Vibrio natriegens / 生物工程学报

Recently, fast-growing Vibrio natriegens, as the great potential chassis, has shown a wide application in synthetic biology. Genome editing is an indispensable tool for genetic modification in synthetic biology. However, genome editing tools with high efficiency and fidelity are still to be developed for V. natriegens synthetic biology. To deal with this problem, the physiological characteristics of 6 V. natriegens strains were evaluated, and CICC 10908 strain with fast and stable growth was selected as the host strain for genome editing study. Then, the natural transformation system of V. natriegens was established and optimized. The efficiencies of optimized natural transformation that integrates antibiotic resistance marker cat-sacB or Kan(R) onto the chromosome of V. natriegens could reach 4×10⁻⁵ and 4×10⁻⁴, respectively. Based on the optimized natural transformation, a double-selection cassette was used to achieve seamless genome editing with high efficiency and fidelity. The positive rates of four different types of genetic manipulation, including gene deletion, complementation, insertion and substitution, were 93.8%, 100%, 95.7% and 100%, respectively. Finally, transformation and elimination of the recombinant plasmid could be easily achieved in V. natriegens. This work provides a seamless genome editing system with high efficiency and fidelity for V. natriegens synthetic biology.

Mechanism and influencing factors of natural transformation in bacteria / 生物工程学报

Horizontal gene transfer contributes to the spread of antibiotic-resistance cassettes, the distribution of toxin-encoding phages and the transfer of pathogenicity islands. Natural transformation, which is the process of competent cells to uptake free DNA from environment and to recombine this DNA into the chromosome, is a mode of horizontal gene transfer. Natural transformation promotes the spread of antibiotic-resistance cassettes among different bacteria, resulting in the emergence of antibiotic resistant bacteria. The emergence of antibiotic resistant pathogens poses an enormous threat to the treatment of infections. Natural transformation could occur in many bacteria, but the mechanism many be different in different bacteria. Also, the inducer and efficiency of natural transformation in different bacteria are influenced by various factors. This review focuses on the mechanism and influencing factors of natural transformation in bacteria.

Construction of psm-negative mutans of S.mutans UA159 / 实用口腔医学杂志

Objective:To knock the phospho-sugar mutase gene (psm) out of the genome of S. mutans strain UA159 and to construct the mutant will lay a foundation for the study of the function of the psm. Methods:Two upstream and downstream DNA sequences of the psm were selected and cloned respectively into multiple cloning sites I and II of suicide plasmid pFW5 to construct the recombinant plasmid which was confirmed by enzyme digesting and sequencing. The recombinant plasmid was transformed into S. mutans UA159 by natural transformation and antibiotic was used to screen the positive transformants. According to the principle of homologous recombination, allelic exchange between the recombinant plasmid and S. mutans UA159 was achieved. Results:By PCR analysis and sequencing, it was confirmed that the psm of S. mutans UA159 was substituted for resistant gene of spectinomycin. Conclusion: The psm-negative mutants of S. mutans UA159 is successfully constructed.