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Development of an effective method to tag escherichia coli chromosomal genes by recombineering

by Leong, Mei-kid

Abstract (Summary)
(Uncorrected OCR) 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.
Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:escherichia coli genetics gene mapping genetic recombination

ISBN:

Date of Publication:01/01/2005

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