Abstract
Red homologous recombination has been extensively used in recombineering. Because foreign sequences, such as antibiotic resistance genes, FRT-sites, or loxP-sites, are often unwanted in mutant Escherichia coli, we established a markerless deletion system containing short homologous sequences, a positive-selectable marker (kan), and a negative-selectable marker (sacB) for E. coli. For markerless deletion of a specific region of the E. coli genome, a two-step recombination procedure using two different PCR fragments, which were amplified from pUC57-kan-sacB and pUC57-298, was performed. The generation of a pheA-tyrA deficient mutant demonstrated that this markerless deletion system was a simple and efficient method to generate markerless chromosomal deletions in E. coli.
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Acknowledgments
This work was supported by the Fundamental Research Funds of Jilin University, the Jilin Province Science and Technology Development Projects (No. 20140101123JC and No. 20150204077NY), Science and Technology Research Program during the 12th Five-year Plan Period of Jilin Educational Committee, and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT1248).
T-sacB plasmid was kindly provided by Dr. Guangmo Yan from Jilin University.
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The authors declare that they have no conflict of interests.
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Fuwang Chen, Jie Jiang and Hongsheng OuYang contributed equally to this work.
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Chen, F., Jiang, J., OuYang, H. et al. Markerless Deletion System for Escherichia coli Using Short Homologous Sequences and Positive–Negative Selectable Cassette. Appl Biochem Biotechnol 176, 1472–1481 (2015). https://doi.org/10.1007/s12010-015-1658-3
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DOI: https://doi.org/10.1007/s12010-015-1658-3