Novel pairs of Bet/Exo recombineering proteins were identified in the beta-proteobacterium Laribacter hongkongensis (LHK) and in the SXT genetic element isolated from Vibrio cholerae. In this research, these new recombineering proteins were functionally characterized using a variety of in vivo and in vitro techniques. The SXT-Exo and LHK-Exo proteins were both found to be alkaline exonucleases, with activities similar to those of Lambda-Exo. Both the SXT-Bet/Exo and LHK-Bet/Exo protein pairs had dsDNA recombination activity within E. coli. / Recombineering is a powerful tool used to manipulate or engineer DNA in vivo, which enables chromosomes and plasmids to be modified precisely and efficiently. It is of critical importance for research into genome and proteome function, and greatly facilitates metabolic engineering applications. The Lambda-Red (Bet and Exo) and RecET proteins constitute the most efficient bacterial recombineering systems characterized to date. However, they work only in E. coli or closely-related bacteria (e.g. Salmonella spp.), which limits their widespread application. / The Lambda-Red and RecET recombineering systems can use PCR products (double stranded DNA, dsDNA) or single stranded DNA (ssDNA, oligonucleotides) to create precise point mutations (substitutions), gene deletions and insertions in chromosomal or episomal DNA. The Exo/RecE exonuclease proteins digest dsDNA and produce long 3'-ssDNA tails. The Bet/RecT ssDNA annealing proteins bind to these 3'-ssDNA tails and promote their homologous recombination with complementary ssDNA regions on the chromosome or episome. / The results described in this thesis will be very useful in assisting the future development of novel recombineering systems that can be used for genetic engineering applications across a wide range of bacterial organisms. Such tools will greatly promote functional genomic and proteomic studies within these organisms, and may also be used for microbial engineering and biotechnological applications. / The ssDNA recombination activities of five different Bet/RecT recombinases were directly compared using an E. coli reporter system. The comparison revealed that Bet protein from LHK had a higher efficiency than Lambda-Bet or RecT. Based on their predicted secondary structure, a set of rationally-designed lambda-Bet protein truncations were prepared and their biological activity was examined, to investigate structure-function relationships within this recombinase. / Chen, Wenyang. / Adviser: Ho W.S. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 113-128). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344703 |
| Date | January 2010 |
| Contributors | Chen, Wenyang., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xiii, 128 leaves : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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