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Development of Genetic Tools for Thermotoga SPP.

Han, Dongmei
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1381840588

Abstract Details

2013, Doctor of Philosophy (Ph.D.), Bowling Green State University, Biological Sciences.
Thermotoga spp. may serve as model systems for understanding life sustainability under hyperthermophilic conditions. They are also attractive candidates for producing biohydrogen in industry. However, a lack of genetic tools has hampered the investigation and application of these organisms. We improved the cultivation method of Thermotoga spp. for preparing and handling Thermotoga solid cultures under aerobic conditions. An embedded method achieved a plating efficiency of ~ 50%, and a soft SVO medium was introduced to bridge isolating single Thermotoga colonies from solid medium to liquid medium. The morphological change of T. neapolitana during the growth process was observed through scanning electron microscopy and transmission electron microscopy. At the early exponential phase, around OD600 0.1 - 0.2, the area of adhered region between toga and cell membrane was the largest, and it was suspected to be the optimal time for DNA uptake in transformation. The capacity of natural transformation was found in T. sp. RQ7, but not in T. maritima. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid isolated from T. sp. RQ7. Plasmid pDH10 was introduced to T. sp. RQ7 by liposome-mediated transformation, electroporation, and natural transformation, and to T. maritima through liposome-mediated transformation and electroporation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extract of the recombinant strains by PCR followed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selection pressure. A uracil auxotrophic strain RQ7-15, with a 115 bp deletion near the 3' end of pyrE gene on T. sp. RQ7 chromosome, was isolated and used as the recipient cell for using pyrE as the selection marker. The pyrE gene from Caldicellulosiruptor saccharolyticus was expressed in RQ7-15, driven by either promoter PslpA from Thermus thermophilus or promoter PRQ7.pyr, which is the promoter of the pyrimidine synthesis operon of T. sp. RQ7. This work advanced the development of genetic tools for the study of Thermotoga.
Zhaohui Xu, Ph.D. (Advisor)
Lisa Chavers, Ph.D. (Committee Member)
George Bullerjahn, Ph.D. (Committee Member)
Raymond Larsen, Ph.D. (Committee Member)
Scott Rogers, Ph.D. (Committee Member)
96 p.
Biology; Microbiology

Recommended Citations

Citations

  • Han, D. (2013). Development of Genetic Tools for Thermotoga SPP. [Doctoral dissertation, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1381840588

    APA Style (7th edition)

  • Han, Dongmei. Development of Genetic Tools for Thermotoga SPP. 2013. Bowling Green State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1381840588.

    MLA Style (8th edition)

  • Han, Dongmei. "Development of Genetic Tools for Thermotoga SPP." Doctoral dissertation, Bowling Green State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1381840588

    Chicago Manual of Style (17th edition)

bgsu1381840588
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© 2013, all rights reserved.
This open access ETD is published by Bowling Green State University and OhioLINK.