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Published online 12 January 2004

Nucleic Acids Research, 2004, Vol. 32, No. 1 e11
© 2004 Oxford University Press

Method to integrate multiple plasmids into the mycobacterial chromosome

Beatrice Saviola* and William R. Bishai12

Basic Medical Sciences, College of Osteopathic Medicine, Western University, 309 E. Second Street, Pomona, CA 91766-1854, USA, 1 Division of Infectious Diseases, Department of Medicine, Johns Hopkins University School of Medicine and 2 Department of International Health, Johns Hopkins School of Hygiene and Public Health, 424 N. Bond Street, Baltimore, MD 21231-1001, USA

*To whom correspondence should be addressed. Tel: +1 909 469 5373; Fax: +1 909 469 5698; Email: bsaviola{at}westernu.edu

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested.


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