Double-strand DNA recombination

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Table of contents

Part I: quinoline biodegradation in soil microcosm
1. Bibliographical review of quinoline and its biodegradation
1.1 Quinoline biodegradation
1.1.1 Pseudomonas sp. for quinoline biodegradation
1.1.2 Rhodococcus sp. for quinoline biodegradation
1.1.3 Comamonas sp. for quinoline biodegradation
1.1.4 Thauera.sp for quinoline biodegradation
1.2 Metabolic quinoline pathway
1.2.1 Metabolic quinoline pathway by Pseudomonas.sp
1.2.2 Metabolic quinoline pathway by Rhodococcus.sp
1.2.3 Metabolic quinoline pathway under anaerobic/anoxic condition
1.3 Genes involved in the quinoline biodegradation
2. Materials and methods
2. 1 Quinoline microcosm
2.2 RISA (rRNA intergenic spacer analysis)
2.3 GC/MS analysis for quinoline biodegradation
3. Results
3.1 Nucleotide BLAST of bcr operon in the Rothamsted metagenome
3.2 RISA results
3.3 Results of GC/MS analysis
4. Discussion and conclusion
PART II: in vitro development and use of Genefish to capture targeted DNA fragments
1. Background
2. Bibliographical review of Lambda-Red homologous recombination and Genefish approach
2.1 Lambda-Red homologous recombination
2. 1.1 Introduction
2.1.2. Lambda-Red recombination system
2.1.2.1 Overview of Lambda-Red recombination system
2.1.2.2 Different vectors for the Lambda-Red system
2.1.2.3 Substrates for the Lambda-Red system
2.1.2.3.1 Double-strand DNA recombination
2.1.2.3.2 Single-strand DNA recombination
β.β. The “Genefish” tool
β.β.1 General presentation of “Genefish”
2.2.2 The suicide cassette
2.2.3 Homologous recombination with Lambda-Red system
2.2.4 bcr operon
3. Genefish application using bcr operon
3.1. Capture plasmid and host strain construction
3.1.1 Materials:
3.1.2 Methods:
3.1.2.1 Highly conserved bcr fragment sequences determination
3.1.2.2 Capture plasmid construction
3.1.2.3 Host strain construction
3.1.2.4 Escape rate test
3.1.3 Results
3.1.3.1 Highly conserved bcr fragment selected for capture plasmid construction
3.1.3.2 Capture plasmid construction
3.1.3.3 Host strain construction and escape rate test
3.2. Genefish tool application
3.2.1 Materials
3.2.2 Methods:
3.2.2.1 Co-electroporation
3.2.2.2 Plasmid segregation
3.2.3 Results.
3.2.3.1 Co-electroporation by using bcr c-d fragment
3.2.3.2 Plasmid segregation by using pBAD35K7toxN-bcrc-d
3.2.3.3 Plasmid segregation by using pBAD35K7toxN-bcrc-a
3.3. Genefish improvements
3.3.1. Materials
3.3.2 Methods:
3.3.2.1 Improvement of co-electroporation and plasmid segregation efficiency through culture time decrease
3.3.2.2 Single-copy plasmid construction
3.3.2.2.1 Classical digestion and ligation
3.3.2.2.2 In-Fusion HD Cloning Kit method
3.3.3 Results:
3.3.3.1 Improved co-electroporation
3.3.3.2 Improved plasmid segregation
3.3.3.3 single-copy capture plasmid construction
4. Conclusions and Discussion
5. Perspectives