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1 Centre for Rhizobium Studies, School of Biological Sciences & Biotechnology, Murdoch University, Perth, Australia
2 Office of the ProViceChancellor (Research), University of Tasmania, Hobart, Tasmania, Australia
ABSTRACT
Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptll-TtrpA) and CAS-Gm (Ptac/P aacCl-aacCl-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptll-TtrpA) or CAS-GGm (gusA- Ptac/PaacCl-aacCl-TtrpA) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-nptll-TtrpA) and mTn5-Gm (containing gusA-Ptac-nptll-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCl-aacCl-T trpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other Gram-negative bacteria.
Author for correspondence: Wayne G. Reeve. Tel: +61 8 9360 2631. Fax: +61 8 9360 6486. e-mail: reeve@possum.murdoch.edu.au
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