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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
[Constructing duplication hasABC of chromosome recombinant in Streptococcus equi subsp. Zooepidemicu]
Xiaoling Lan1, Bo Zhang, Xueru Li
1School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China.
Objective:
To investigate the possibility of increasing the yield of hyaluronic acid by constructing duplication hasABC of chromosome recombinant in Streptococcus equi subsp. Zooepidemicus with a thermosensitive delivery vector system pJR700.
Methods:
We amplified a 4147 bp DNA fragment of hyaluronic acid synthase operon hasABC genes from chromatosome of S. zooepidemicus using PCR. This DNA fragment was subcloned into the pJR700 at ClaI sites to result in recombinant plasmid pXL32. The recombinant plasmid was transformed into S. Zooepidemicus by electroperation. The homologous recombination was induced by growing the bacteria at 37 degrees C, and transformants were selected according to kanamycin resistance for 3 rounds. Then the culture was shifted to grow at 30 degrees C without antibiotics for 4 rounds to induce excision of the pJR700 indicated fragment. Colonies with kanamycin sensitivity were selected by plating on THY agar at 37 degrees C. The hasABC recombinant of S. Zooepidemicus was identified through RT-PCR with primers homologous to the flanking regions. HA titers were measured by the modified carbazole assay.
Results:
We constructed successfully the duplication hasABC of chromosome recombinant of S. Zooepidemicus and the HA titer production by recombinants harboring duplication hasABC was 34% higher than that of the wild type at 24 h in shake flask culture.
Conclusion:
The thermosensitive delivery vector of pJR700 could be used to construct the streptococcal hasABC recombinant strain for increasing the yield of HA in S. Zooepidemicus.
