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Summer Research Fellowship Programme of India's Science Academies

Fluorescent tagging of Bradyrhizobium sp. DOA9 and its two mutants DOA9 delnodB, DOA9 delrhcN

Pankaj Pandey

Bachelor of Biotechnology, National Institute of Technology Sikkim, Sikkim 737139

Prof. Maitrayee DasGupta

Department of Biochemistry, University of Calcutta, Kolkata 700019

Abstract

Bacterial colonization in plants can be studied best once the colonizing microbes are tagged fluorescently. Arachis hypogaea is a legumes plant which can form nodules and fix Nitrogen with diverse Bradyrhizobium. Bradyrhizobium is a difficult genera to work with when it comes to labeling of the various strain members. The Tn7 system has proved to be effective in fluorescently tagging various bacterial species, including Pseudomonas. The Tn7 system for labeling of bacteria involves integration of Tn7 transposon-flanked portion of the vector within the chromosome of Bradyrhizobia having a single attTn7 site. Following labeling, confirmation of the insertion is done via molecular biology techniques like PCR. In this project, we have employed the Tn7 system for labeling Bradyrhizobium DOA9 and its two mutants delrhcN and delnodB. The procedure involves, first, cloning of the genes of interest into an appropriate mini-Tn7 vector; second, co-transfer of the recombinant mini-Tn7 vector and a helper plasmid encoding the Tn7 site-specific transposition pathway into Bradyrhizobium DOA9 and its mutant delnodB and delrhcN by conjugation, followed by selection of insertion-containing strains; third, PCR verification of mini-Tn7 insertions. After verifying the presence of Tn7 system in recipient bacteria, they are used to infect Arachis hypogaea in order to study the colonization pattern.

Keywords Bradyrhizobium sp DOA9, Tn7 System, Leguminious Plant, Transpotion,

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