Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise replacement of the pyrF gene

Plasmid
N KnipferT E Shrader

Abstract

After integration into the bacterial chromosome an exogenous gene may be stably expressed without continued selection for the recombinant locus. However, chromosomal integration events occur infrequently, requiring the concomitant integration of a drug resistance marker in order to identify colonies of recombinant cells. The generation of a drug-resistant recombinant strain can both reduce the in vivo applicability of the strain and preclude the use of recombinant vectors which use the same drug resistance marker. We have constructed a plasmid, pINT-delta, which allows recombination of exogenous genes onto the Mycobacterium smegmatis chromosome. The exogenous gene completely replaces the pyrF gene and the resultant strain lacks any exogenous drug resistance marker. The methodologies described herein are general and applicable even to those bacteria for which extrachromosomal plasmids are not available. Using pINT-delta we integrated the lacZ gene into the M. smegmatis chromosome via a precise exchange of lacZ and pyrF. The resultant strain was used to demonstrate that the expression of genes integrated at the pyrF locus is repressed twofold by inclusion of uracil in the growth medium. In addition, we used pINT-delta to construc...Continue Reading

References

Jan 11, 1991·Methods in Enzymology·W R JacobsB R Bloom
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Jun 1, 1995·Molecular Microbiology·P SanderE C Böttger
Nov 1, 1993·Journal of Bacteriology·A AldoviniR A Young

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Citations

Feb 5, 2005·Applied and Environmental Microbiology·Teca Calcagno Galvão, Víctor de Lorenzo
Dec 26, 2006·FEMS Microbiology Letters·Ricardo F CaponeJ Christopher Fenno
Dec 20, 2015·Applied and Environmental Microbiology·Kurni Kurniyati, Chunhao Li

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