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Development of an Effective Method to Tag Escherichia Coli Chromosomal Genes by Recombineering

Development of an Effective Method to Tag Escherichia Coli Chromosomal Genes by Recombineering


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About the Book

This dissertation, "Development of an Effective Method to Tag Escherichia Coli Chromosomal Genes by Recombineering" by Mei-kid, Leong, 梁美潔, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled DEVELOPMENT OF AN EFFECTIVE METHOD TO TAG ESCHERICHIA COLI CHROMOSOMAL GENES BY RECOMBINEERING Submitted by Mei Kid Leong for the Degree of Doctor of Philosophy at the University of Hong Kong in July 2004 The methods currently used to tag E. coli proteins are time consuming, and the efficiency of the tagging process is low. Consequently, the development of efficient tagging protocols are required to accommodate the ever increasing demands of high throughput proteomic studies in bacteria, as well as in eukaryotic organisms. To address this problem, a strategy for large-scale genome tagging was developed, using Escherichia coli as our model organism. A set of template plasmids, incorporating nine different fluorescent and one tandem affinity purification (TAP) gene-fusions were constructed, suitable for tagging all the open reading frames (ORFs) in the E. coli genome. A representative selection of E. coli genes were tagged using these templates, utilizing new combinations of recombineering technologies; involving site- specific and homologous recombination-based procedures. The fluorescently tagged proteins were dynamically-localized within the cell using fluorescence and confocal microscopy. These endogenously-expressed tagged proteins exhibited a variety of different intracellular locations: some distributing throughout the cytoplasm, some adhering to the membranes and others forming a number of discrete foci. The TAP- tags were used to capture and isolate protein complexes through a gentle, yet highly stringent, two-step affinity chromatography procedure. The work contained within this thesis provides a solid platform from which further large-scale proteomic and genomic studies may be launched. DOI: 10.5353/th_b3043081 Subjects: Escherichia coli - Genetics Gene mapping Genetic recombination


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Product Details
  • ISBN-13: 9781374720862
  • Publisher: Open Dissertation Press
  • Publisher Imprint: Open Dissertation Press
  • Height: 279 mm
  • No of Pages: 166
  • Weight: 399 gr
  • ISBN-10: 1374720860
  • Publisher Date: 27 Jan 2017
  • Binding: Paperback
  • Language: English
  • Spine Width: 9 mm
  • Width: 216 mm


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