15A-5 |
Construction of a food-grade cloning vector based on a small lactococcal cryptic plasmid, pCL2.1 |
S. H. CHO1, J. M. Lee1, J. H. Kim2, and H. J. Lee1. (1) Dept. of Food Science & Technology, Seoul National Univ., School of Agricultural Biotechnology, Suwon, 441-744, South Korea, (2) Dept. of Food Science & Technology, Gyeongsang National Univ., Chinju, 660-701, South Korea Lactic acid bacteria (LAB) are widely utilized as starters for various food fermentations. As new applications of LAB emerge, need for strain improvement through genetic engineering also increases. Food-grade cloning vectors are valuable tools for the production of desirable metabolites, enzymes, and other proteins. However, all the developed vector systems have certain limitations in terms of host range and effectiveness of selection markers, justifying further efforts to develop novel vectors. In our previous studies, pCL2.1, the smallest (2.1 kb in size) resident plasmid of Lactococcus lactis ssp. lactis ML8, was characterized. Our objective was to construct a food-grade cloning vector based on pCL2.1 as a vector frame and origin with a food-grade selection marker, b-galactosidase gene (lacZ) cloned from Lactococcus lactis ssp. lactis ATCC 7962 and a lactococcal promoter. Molecular cloning techniques and plasmid DNA isolations were performed as described by Sambrook et al. L. lactis electroporations were carried out as described by Holo and Nes. First, pCL2.1 was cleaved with ClaI and ligated with AccI-digested pUC18, resulting in a 4.8 kb chimeric plasmid, pUCL2.1. Then erythromycin resistance gene (Emr) was introduced into pUCL2.1 as a temporary selection marker for LAB. The lacZ and multiple cloning site (MCS) was introduced into pUCL2.1. Also, a lactococcal promoter sequence, p13c isolated from L. lactis chromosome was introduced upstream of MCS. After these manipulations, Emr marker and other sequences originated from E. coli were removed. The new food-grade cloning vector was named as pSH110 and introduced into L. lactis ssp. cremoris LM0230 and MG1363 by electroporation. On X-gal plates, blue colonies appeared and the presence of pSH110 was confirmed. pSH110 was stably maintained in these L. lactis strains as long as grown on lactose. These results suggest that the pSH110 could be used as a new food-grade cloning vector in LAB.
Session 15A, Biotechnology
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