Food-grade Cloning Research Articles

Development and Application of pFM011 as a Possible Food-Grade Cloning Vector

An origin of replication and a nisin resistance determinant on a 7.6-kb EcoRI fragment, capable of existing as an independent replicon when circularized, was used to construct a vector for cloning homologous DNA in Lactococcus lactis ssp. lactis. A medium, designated M17-GTN, was developed to identify L. lactis ssp. lactis cells containing pFM011 from a mixed population of nisin-sensitive resistant transformants produced by electroporation or by protoplast transformation. To demonstrate the usefulness of nisin resistance as a selectable marker, a 6.3-kb EcoRI fragment encoding reduced bacteriophage sensitivity was cloned into the EcoRI site of pFM011. Separately, the erythromycin resistance marker from pGB301 was cloned into the HindIII site of pFM011. Both plasmids were introduced into L. lactis. ssp. lactis. The origin of replication of pFM011 was localized to a 2.2-kb HindIII-HaeIII fragment. Seven additional unique restriction sites added by inserting a multiple cloning site. Results indicated that pFM011 and its recombinant derivatives were relatively stable in the L. lactis ssp. lactis background. As pFM011 was derived entirely from lactococcal DNA, this study demonstrates its use as a food-grade cloning vector for lactococci.

Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli.

The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region.