钱宏亮经验交流ISceI.deletion.stepbystep.manual.v2.ppt

Homing Endonucleases I-SceI Mediated Markerless Gene Replacement in Bacillus thuringiensis str.BMB171 Gene replacement strategy Homologous recombination Plasmid elimination Screening The second plasmid which contains the gene encoded the I-SceI enzyme was introduced by electroporation. The I-SceI cleavaging at the unique site within the vector resulted in chromosomal double-stranded break, which can sitimulated the host repair system to repair the DSB by homologous recombinatjion between flanking repeat sequences leads to plasmid excision, in which approximately 50% will have undergone the desired gene knock-out. The first plasmid that contains the homologous fragment of the upstream and downstream region of the target gene is introduced into the Bt. BMB171 by conjugative transfer . Then the recombinants that the whole pRP1028-Bud plasmid integrates into the Bt. BMB171 chromosome by homologous recombination was isolated by shifting them to 37 oC. The first plasmid that contains the homologous fragment of the upstream and downstream region of the target gene is introduced into the Bt. BMB171 by conjugative transfer . Then the recombinants that the whole pRP1028-Bud plasmid integrates into the Bt. BMB171 chromosome by homologous recombination was isolated by shifting them to 37 oC. The second plasmid which contains the gene encoded the I-SceI enzyme was introduced by electroporation. The I-SceI cleavaging at the unique site within the vector resulted in chromosomal double-stranded break, which can sitimulated the host repair system to repair the DSB by homologous recombination between flanking repeat sequences leads to plasmid excision, in which approximately 50% will have undergone the desired gene knockout. Homing Endonucleases Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes init