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URN etd-0218108-163156 Statistics This thesis had been viewed 5409 times. Download 5936 times. Author Shiao-Ming Chang Author's Email Address No Public. Department Bioengineering Year 2007 Semester 1 Degree Ph.D. Type of Document Doctoral Dissertation Language zh-TW.Big5 Chinese Page Count 183 Title Screen of Probiotic Lactic Acid Bacteria and Constructing Shuttle Vector of Lactic Acid Bacteria Keyword shuttle vector system Lactic acid bacteria Probiotics pL2 plasmid theta-type replicating plasmid theta-type replicating plasmid pL2 plasmid Probiotics Lactic acid bacteria shuttle vector system Abstract The results are divided into two parts with screen of probiotic Lactic acid bacteria and constructing shuttle vector of Lactic acid bacteria. In part I, the probiotic Lactic acid bacteria (LAB) was screened and used for provide intestinal health and host of shuttle vector of LAB. In part II, a cryptic LAB plasmid was isolated and the plasmid sequence was determined. Based on LAB plasmid, a shuttle vector was constructed, it could maintain stably in probiotic LAB and express heterologous protein gene. In screen of probiotic LAB, the twenty-one of LAB strains which could withstand high acid and bile salt conditions were selected. These strains meanwhile could tolerate digestive system, restrain pathogenic bacteria growth, metabolize isomaltooligosaccharides and gentiooligosaccharides, and induce cytokine production (IL-12 p40p70 and IL-10) by RAW 264.7 macrophage cell. All of these strains could be used as a candidate for probiotics. The species were identified as Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus by API and 16S rRNA. Strain E1, E33, E40, and E55 were plasmid free and selected as the host of shuttle vector system of LAB. In constructing shuttle vector of LAB, a cryptic plasmid pL2 was isolated from Lactococcus lactis subsp. lactis and its complete nucleotide sequence was determined (5,299-bp, GenBank accession No. DQ917780). Its replication mode was identified as a theta-type belonging to the pAMβ1 family. Analysis of the nucleotide sequence revealed that pL2 contained a transfer origin, a replication origin, and five putative open reading frames (ORF 1-5). ORF1 (386 amino acids) was homologous to replication protein RepB. The shuttle vector pUL6erm was constructed by using a replicon from pL2, a multiple cloning site, colE1 ori, the origin of Gram-negative bacteria from vector pUC19, and the erythromycin resistance gene from pVA838 as a selective marker. The pUL6erm could be transformed easily and maintained stably in E. coli, Lactobacillus casei, Lactobacillus plantarum, Lactococcus lactis, and Streptococcus thermophilus. The expression plasmid pUL6erm- gadR-GUS was constructed base on pUL6erm and a chloride-inducible gene expression cassette encoding gadR and the Pgad promoter. Growth in the presence of 0.3 M sodium chloride and 50 mM glutmate, the beta-D-glucuronidase was induced and expressed with 2.37 Unit/mg by plasmid pUL6erm-gadR-GUS. Advisor Committee Tsong-Rong Yan - advisor
C. Will Chen - co-chair
Chorng-Liang Pan - co-chair
Kow-Jen Duan - co-chair
Roch-Chui Yu - co-chair
Ying-Chieh Tsai - co-chair
Files Date of Defense 2008-01-11 Date of Submission 2008-02-19