Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti

Pobigaylo N, Wetter D, Szymczak S, Schiller U, Kurtz S, Meyer F, Nattkemper TW, Becker A (2006)
APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72(6): 4329-4337.

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Sinorhizobium meliloti genome sequence determination has provided the basis for different approaches of functional genomics for this symbiotic nitrogen-fixing alpha-proteobacterium. One of these approaches is gene disruption with subsequent analysis of mutant phenotypes. This method is efficient for single genes; however, it is laborious and time-consuming if it is used on a large scale. Here, we used a signature-tagged transposon mutagenesis method that allowed analysis of the survival and competitiveness of many mutants in a single experiment. A novel set of signature tags characterized by similar melting temperatures and G+C contents of the tag sequences was developed. The efficiencies of amplification of all tags were expected to be similar. Thus, no preselection of the tags was necessary to create a library of 412 signature-tagged transposons. To achieve high specificity of tag detection, each transposon was bar coded by two signature tags. In order to generate defined, nonredundant sets of signature-tagged S. meliloti mutants for subsequent experiments, 12,000 mutants were constructed, and insertion sites for more than 5,000 mutants were determined. One set consisting of 378 mutants was used in a validation experiment to identify mutants showing altered growth patterns.
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Pobigaylo N, Wetter D, Szymczak S, et al. Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2006;72(6):4329-4337.
Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., Nattkemper, T. W., et al. (2006). Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72(6), 4329-4337.
Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., Nattkemper, T. W., and Becker, A. (2006). Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72, 4329-4337.
Pobigaylo, N., et al., 2006. Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 72(6), p 4329-4337.
N. Pobigaylo, et al., “Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti”, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 72, 2006, pp. 4329-4337.
Pobigaylo, N., Wetter, D., Szymczak, S., Schiller, U., Kurtz, S., Meyer, F., Nattkemper, T.W., Becker, A.: Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 72, 4329-4337 (2006).
Pobigaylo, N, Wetter, D, Szymczak, S, Schiller, U, Kurtz, S, Meyer, F, Nattkemper, Tim Wilhelm, and Becker, A. “Construction of a large signature-tagged mini-Tn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti”. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 72.6 (2006): 4329-4337.
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