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Appl Environ Microbiol. 2006 Jun;72(6):4329-37.

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

Author information

1
Lehrstuhl f├╝r Genetik, Universit├Ąt Bielefeld, Postfach 100131, 33501 Bielefeld, Germany.

Abstract

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.

PMID:
16751548
PMCID:
PMC1489598
DOI:
10.1128/AEM.03072-05
[Indexed for MEDLINE]
Free PMC Article
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