How are we going to knockout the FadR gene? To do that, we choose two alternatives: the first is gene interruption for homologous recombination and the second is deleted the FadR gene using a two-step asymmetric/crossover PCR amplification. (1) We design primers to the internal region of FadR gene and after the amplification we insert this gene in a TOPO vector, cloning in E. coli RR1, miniprep the plasmids, digest with ApaI and BamHI and ligation the insert in pKNOCK and pNPT138 plasmids digested tih ApaI and BamHI.
Fig 1. Design of pKNOCK Integrative vector with FadR internal region. And these plasmid will be transformed in Shewanella putrefaciens to occur homologous recombination and the FadR gene be interruption by vector integration. (2) Design PCR primers to amplify the regions flanking the gene FadR. Subsequent to amplification, these flanking regions are fused together via a complementary “tag” region. This fusion product will then be inserted into the suicide vector pNPTS138.
Fig 2. Design of pNPTS138 vector with flanking genes This plasmid will be transform in Shewanella putrefaciens and the suicide plasmid will Integrate on genomic DNA during a single crossover event.
Fig 3. Integration of the suicide plasmid during a single crossover event. Depending on whether the recombination occurred between the 5’ fragments or the 3’ fragments, you will obtain one of the above insertion patterns (A or B). And then we will force a excision of the suicide plasmid by cultive in extress médium (Lb with 5% of sucarose, without NaCl and antibiotcs) turn deleted gene copy strain or wild type
Figure 4. Generation open-frame deletion during the excision of the suicide plasmid.