Development of food-grade cloning and expression vectors for Lactococcus lactis.
OBJECTIVE
To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis.


RESULTS
Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis.


CONCLUSIONS
The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis.


CONCLUSIONS
These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.
Liu
C-Q
CQ
Department of Biotechnology, University of New South Wales, Sydney, Australia. chun.liu@dsto.defence.gov.au
Su
P
P
Khunajakr
N
N
Deng
Y-M
YM
Sumual
S
S
Kim
W S
WS
Tandianus
J E
JE
Dunn
N W
NW
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D005516:Food Microbiology; D015870:Gene Expression; D005819:Genetic Markers; D005822:Genetic Vectors; D013294:Lactococcus lactis / Q000235:genetics*; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D011401:Promoter Regions, Genetic
D005819:Genetic Markers; D004247:DNA