In:
Microbial Biotechnology, Wiley, Vol. 8, No. 1 ( 2015-01), p. 177-187
Abstract:
Chromosomal integration of expression modules for transgenes is an important aspect for the development of novel S almonella vectors. Mini‐ Tn 7 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‐ Tn 7 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‐ Tn 7 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‐ R ed 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 Tn 7 target immunity. Integration of multiple copies of a mini‐ Tn 7 containing a gfp cassette resulted in increasing green fluorescence of bacteria. Stable consecutive integration of two mini‐ Tn 7 encoding lacZ and lux was achieved by initial transposition of lacZ ‐mini‐ Tn 7, subsequent chromosomal insertion of a ‐ attTn 7 and a second round of transposition with lux ‐mini‐ Tn 7. Mini‐ Tn 7 thus constitutes a versatile method for multicopy integration of expression cassettes into the chromosome of S almonella and possibly other bacteria.
Type of Medium:
Online Resource
ISSN:
1751-7915
,
1751-7915
DOI:
10.1111/mbt2.2015.8.issue-1
DOI:
10.1111/1751-7915.12187
Language:
English
Publisher:
Wiley
Publication Date:
2015
detail.hit.zdb_id:
2406063-X
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