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| Optimization of the Method for Scarless Gene Knockout in Escherichia coli Genome |
| GE Gao-shun1, ZHANG Li-chao1, ZHAO Xin2, HU Xue-jun1, LI Ya-jie1 |
1. Medical College, Dalian University, Dalian 116622, China;
2. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China |
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Abstract Objective: The purpose was to improve the efficiency of scarless gene knockout in E. coli genome by the optimized method. Methods: The efficacy of scarless gene knockout in E.coli genome by two steps Red homologous recombinantion system and endonuclease I-SceI screening was investigated via optimization of the homologous DNA length with target sequence and the inducer concentration for production of I-SceI for the selection of positive clones. The scarless knockout of nanKETA clusters in the strain CLM37 was taken as a model. The new strain growth behaviour with the scarless knockout of nanKETA was investigated via the comparing the growth curves of wildtype E. coli CLM37. Results: The nanKETA clusters were knockouted scarless successfully in E. coli CLM37 genome, and the efficacy of scarless processing was increased up to 90% via extending the length of the homologous DNA with the genome, from the 80 base pairs normally used up to 684 base pairs, and increasing the concentration of the inducer tetracycline for producing I-SceI, from 500 μg/ml upto 1000 μg/ml. It is shown that the knockout of nanKETA clusters in E. coli CLM37 did not impact the growth. Conclusion: The efficacy of gene scarless knockout in E.coli can be significantly improved via extending the length of the homology DNA and increasing the concentration of inducer tetracycline.
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Received: 05 March 2014
Published: 25 June 2014
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