Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi |
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Authors: | Eom Hyun-Ju Moon Jin-Seok Cho Seung Kee Kim Jeong Hwan Han Nam Soo |
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Institution: | a Department of Food Science and Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea b Division of Applied Life Science, Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea |
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Abstract: | The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312 bp, a low G + C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the β-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB. |
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Keywords: | Leuconostoc citreum CB2567 Cryptic plasmid Shuttle vector Electroporation qPCR Protein expression |
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