Therefore, the generated mutant can be readily screened on an agar plate containing sucrose. Figure 1 Plasmids constructed to introduce an unmarked mutation into a large gene of non-competent bacteria. (A, B) Multiple cloning VS-4718 cell line sites (MCS) of pJQ200sk and pK18mob were substituted with that of pLOI2224, generating pJQFRT and pKFRT, respectively. The pJQFRT plasmid contains a single FRT site adjacent to a multiple
cloning site; p15A origin, a replication origin of E. coli; oriT, origin of transfer; SacB, a counter-selection marker; and GmR, a gentamicin resistance marker. The arrows indicate the primers used in PCR to amplify the substitute MCS. The nucleotide sequences of these primers are shown in Table 2. (C) A cassette containing tetR-Ptet
promoter and flp recombinase amplified by PCR from pFT-A was ligated with the inverse-PCR product of pKFRT. The resultant pKFRT/FLP plasmid contains a single FRT site adjacent to a multiple cloning site; TetR-FLP, flp recombinase gene under the control of the tetR regulation system; KmR, a kanamycin resistance marker; oriT, origin of transfer; and ColE1 origin, a replication origin of E. coli. Figure 2 Scheme for the unmarked deletion of a large gene by FLP/FRT recombination. The plasmid pJQFRT with the insertion of the upstream region of the target Autophagy inhibitor gene is integrated into the host chromosome by homologous recombination. Next, the plasmid pKFRT/FLP with the insertion of the downstream region of the target gene is integrated into the host chromosome by homologous recombination. As a result, the target gene is sandwiched between the two integrated plasmids. The expression of flp is induced by adding anhydrotetracycline, and then the target region is excised together with the integrated plasmids bracketed by the two FRT sites, leaving a single FRT site. In our methodology, the new gene replacement plasmids pJQFRT and pKFRT/FLP are used for introducing
the unmarked mutation. Since these plasmids are mobilized by bacterial conjugation, there is no concern about the nucleolytic degradation of the introduced plasmid DNA, unlike linear DNA. Loperamide Besides, flp recombinase is cloned under the regulation of the tet promoter in pKFRT/FLP and is integrated into the chromosome of the recipient strain after homologous recombination. Therefore, our method obviates the need for helper plasmids expressing FLP recombinase and λ Red recombinase, which prevents degradation of the introduced linear DNA . Our method can be used in various species of Gram-negative bacteria except for E. coli and some enterobacteria, independent of their competency and recombination ability. Implementation of the new method for the deletion of a large gene from the Acinetobacter sp.