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Minerva Biotecnologica 2012 December;24(4):141-54


lingua: Inglese

Optimization of the λ Red recombinase system for disruption of virG in Shigella dysenteriae type 1 by using the Taguchi design method

Heiat M., Saadati M., Kooshki H., Cheraghi S., Hosseini S. M.

Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran


Shigella Spp. continues to be a major cause of acute diarrheal illness in young children and infants in less-developed countries. Since protective immunity against shigellosis is serotype specific, it seems that construction of a live Shigella dysenteriae type 1 strain attenuated by deletion of the virG gene, a significant virulence gene in pathogeneticity, is urgently needed. To construct a live attenuated Shigella dysenteriae type 1, the polymerase chain reaction (PCR) fragment carrying a chloramphenicol resistance cassette flanked by short (46 nt) regions homologous to the virG gene was electroporated into recipient Shigella dysenteriae strains to express a highly proficient gβexo gene system. The PCR and sequencing analysis demonstrated 3220-nt of virG gene was successfully deleted. To intensify recombination efficiency, however, we used 232 ng of purified PCR-product. The percentage of recombination efficiency was determined to be about 76%. Finally, in order to optimize and improve homologue recombination, we used a new approach in the field of λ Red recombinase optimization by applying an experimental design called the Taguchi method. This method based on a two level, four variable L8 orthogonal array design was implemented to optimize experimental parameters. The effects of factors including arabinose concentration media, concentration of antibiotic in plating media, temperature after electroporation and length of homology arms on the percentage yield of mutant clones were investigated. The optimum conditions derived from using the Taguchi method were: arabinose concentration media 0.1mM, temperature after electroporation 37 °C, concentration of antibiotic in plating media 5 μg, and length of homology arms 475. The actual experimental yield was 202 CFU/mL under optimum and the actual yield at optimum conditions was 15 CFU/mL.

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