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Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici

Liao, Yu-Hua
http://rave.ohiolink.edu/etdc/view?acc_num=osu1072778140

Abstract Details

2004, Master of Science, Ohio State University, Food Science and Nutrition.
Currently, the U.S. market for propionate is about 400 million pounds per year, and the demand is increasing at 3 ~ 4 % annually. Fermentation process can use byproducts from the food industry, such as corn steep liquor and cheese whey, to produce propionic acid. It is thus desirable to convert these low-grade byproducts to a marketable product, such as propionic acid by fermentation. However, propionic acid fermentation with Propionibacterium acidipropionici is limited by the low product yield due to its heterofermetation nature. Metabolic engineering to modify the dicarboxylic acid pathway can be an effective method to alter carbon flow to improve propionate yield by knocking-out the acetate formation. Acetate kinase (ACK) is a key enzyme involving acetate formation in the pathway, and was the knockout target in this research. Integrational mutagenesis by homologous recombination can be used to disrupt ack gene by first constructing a non-replicative plasmid with partial ack and then transforming it into cells. Integration of the non-replicative plasmid can result in duplicate repeats of the homologous regions flanking DNA sequences, and thus cause mutagenesis. In this work, partial ack, encoding ACK, was first cloned and sequenced by PCR based cloning. A 477 bp of partial ack sequence was obtained. However, low yield of genomic DNA preparation from P. acidipropionici might have attributed to failure to clone the ack gene from the genomic DNA using the degenerate primers in the PCR. A selective marker, TetR, failed to ligate into plasmid with partial ack due to the difficulty of ligating two large DNA fragments. Further study to improve the cloning method is necessary to construct the plasmid needed for transformation
SHANG-TIAN YANG (Advisor)
PCR; cloning; acetate kinase; Propionibacterium acidipropionici

Recommended Citations

Citations

  • Liao, Y.-H. (2004). Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1072778140

    APA Style (7th edition)

  • Liao, Yu-Hua. Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici. 2004. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1072778140.

    MLA Style (8th edition)

  • Liao, Yu-Hua. "Polymerase chain reaction based cloning of acetate kinase in propionibacterium acidipropionici." Master's thesis, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1072778140

    Chicago Manual of Style (17th edition)

osu1072778140
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© 2004, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.