Developing a temperature-sensitive plasmid to create Brucella knockout mutants
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Gene knockouts in an organism give valuable information about that gene's function. The current method for producing Brucella knockout mutants - sacB insertion followed by sucrose counterselection - is inefficient. A novel procedure for generating unmarked Brucella knockouts is proposed. First, the target gene is replaced by an antibiotic resistance marker via homologous recombination. Then, this marker is excised by flippase, an enzyme encoded by a temperature-sensitive plasmid. The plasmid is subsequently removed from Brucella by raising the temperature. To obtain such a temperature-sensitive mutant, the broad-host-range plasmid pBBR1 was mutated using the E. coli strain, XL1-Red. Mutation of the plasmid DNA was verified by blue/white screening. Afterwards, the mutants were screened for temperature-sensitivity by replica plating and growth at the permissive (30°C) and non-permissive (42°C) temperatures. A total of 2,400 colonies were screened, and two temperature-sensitive plasmids were isolated. However, further analysis by replica plating showed that the two plasmids were temperature-sensitive for antibiotic resistance, not replication. These plasmids cannot be removed from Brucella by raising the temperature. Hence, it is not yet clear whether the proposed method can generate knockout mutants of Brucella. Future studies should mutate pBBR1 more extensively, or apply the mutagenesis procedure to a different plasmid, such as pGL10.
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Includes bibliographical references (leaves 17-18).
Farooqi, Midhat Saleem (2004). Developing a temperature-sensitive plasmid to create Brucella knockout mutants. Texas A&M University. Available electronically from