A Rapid Method for Constructing Multiply Marked Strains of Bacillus subtilis

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Abstract
B. subtilis mutant 91 (spoVB91 trpC2) was transformed with spoVB+ phe-12 DNA (from strain 34.1), the culture was given the opportunity to sporulate then heat-treated, and survivors were identified. Approximately equal numbers of Spo+ and Spo- colonies were obtained. No survivors of either type were obtained when the DNA was previously degraded by DNAase, when DNA carrying the spoVB91 mutation was used or when no DNA was used. Of 88 Spo- transformants tested, 10 were phenylalanine auxotrophs. One of these, SL11, was used to build up a series of multiply marked auxotrophs. In this way, in 4 successive transformations up to 6 distinct auxotrophic mutations were introduced into strain 91 without loss of the original spoVB91 and trpC2 mutations. Should a Spo+ strain be required, the spoVB91 mutation can be readily removed by an additional transformation. Alternatively, in a series of crosses building up a particular strain, the Spo+ transformant could be tested for the appropriate auxotrophic requirements at the final stage.