1. Construction of a shuttle vector based on the small cryptic plasmid pJY33 from Weissella cibaria 33.
- Author
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Park, Ji Yeong, Jeong, Seon-Ju, Sa, Hyun Deok, Lee, Jae Yong, Liu, Xiaoming, Cho, Min Jeong, Lee, Kang Wook, and Kim, Jeong Hwan
- Subjects
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PLASMIDS , *OPEN reading frames (Genetics) , *GENETIC code , *ESCHERICHIA coli , *LEUCONOSTOC mesenteroides , *GALACTOSIDASES - Abstract
A cryptic plasmid, pJY33, from Weissella cibaria 33 was characterized. pJY33 was 2365 bp in size with a GC content of 41.27% and contained two putative open reading frames (ORFs). orf1 encoded a putative hypothetical protein of 134 amino acids. orf2 was 849 bp in size, and its putative translation product exhibited 87% identity with a replication initiation factor from a plasmid from W. cibaria KLC140. A Weissella - Escherichia coli shuttle vector, pJY33E (6.5 kb, Em r ), was constructed by ligation of pJY33 with pBluescript II SK(−) and an erythromycin resistance gene (Em r ). pJY33E replicated in Lactococcus lactis , Leuconostoc citreum , Lactobacillus brevis, Lactobacillus plantarum, and Weissella confusa . A single-stranded DNA intermediate was detected from Lb. brevis 2.14 harbouring pJY33E, providing evidence for rolling-circle replication of pJY33. Most Lb. brevis 2.14 cells (85.9%) retained pJY33E after one week of daily culturing in MRS broth without Em. An aga gene encoding α-galactosidase (α-Gal) from Leuconostoc mesenteroides was successfully expressed in Lb. brevis 2.14 using pJY33E, and the highest level of α-Gal activity (36.13 U/mg protein) was observed when cells were grown on melibiose. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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