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through Sustainable Agriculture. © Springer Science + Business Media B.V. 2008 F. D. Dakora et al. (eds.), Biological Nitrogen Fixation: Towards Poverty Alleviation 293 To investigate gene function, we developed a robust transcription fusion reporter vector to measure gene expression in bacteria. The vector, pTH1522, was used to construct a random insert library for the Sinorhizobium meliloti genome (Cowie et al., 2006). Homologous recombination of the DNA fragments cloned in pTH1522 into the S. meliloti genome generates transcriptional fusions to either the reporter genes gfp+ and lacZ or gusA and rf p , depending on the orientation of the cloned fragment. Over 12,000 fusion junctions in 6,298 clones were identified by DNA sequence analysis and the plasmid clones were recombined into S. meliloti. Reporter enzyme activities following growth of these recombinants in complex media (LBmc) and in minimal medium with glucose or succinate as sole carbon sources, allowed the identification of genes highly expressed in one or more growth conditions and those expressed at very low to background levels. |
