부산대학교 도서관

Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel S almonella vectors. Mini- Tn7 transposons have been used for the insertion of one such module into the chromosomal site attTn 7, present only once in most Gram-negative bacteria. However, integration of multiple mini- Tn7 copies might be suitable for expression of appropriate amounts of antigen or combination of different modules. Here we demonstrate that integration of a 9.6 kb mini- Tn7 harbouring the luciferase luxCDABE ( lux) occurs at the natural attTn 7 site and simultaneously other locations of the S almonella chromosome, which were engineered using λ- Red recombinase to contain one or two additional artificial attTn 7 sites ( a - attTn 7). Multicopy integration even at closely spaced attTn 7 sites was unexpected in light of the previously reported distance-dependent Tn7 target immunity. Integration of multiple copies of a mini- Tn7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini- Tn7 encoding lacZ and lux was achieved by initial transposition of lacZ-mini- Tn7, subsequent chromosomal insertion of a - attTn 7 and a second round of transposition with lux-mini- Tn7. Mini- Tn7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of S almonella and possibly other bacteria. [ABSTRACT FROM AUTHOR]