Introducing specific mutations into the Escherichia coli chromosome using linear transformation

In 1885 Theodor Escherich described the gram negative bacterium Escherichia coli (E. coli)
(Escherich T., Rev.1989). The gram negative rod belongs to the family of the
enterobacteriaceae. It is a natural inhabitant of the human and animal intestine. E. coli can
also cause diseases like diarrhea, inflammation of the urinary tract or the gall bladder. Urinary tract infections (UTIs) are one of the most common reasons for antibiotic therapy
worldwide (Burman and Olsson-Liljequist, 2001). People with UTIs suffer inflammation of
the urinary tract, and frequently the kidneys. Two-thirds of patients with UTIs are women
(Canbaz et al. 2002). This is related in part to the shortness of the urethra, which makes
colonization of the bladder by bacteria more likely. The elderly and those who undergo
genitourinary operations and catheterisation are also frequent sufferers of UTIs (Orenstein and
Wong, 1999). The leading causative agent of UTIs is E. coli (60-80 %), usually originating
from the patients own faecal flora, followed by Staphylococcus saprophyticus (10 %),
Klebsiella sp., other Gram negative bacteria and enterococci (Burman and Olsson-Liljequist, 2001). The antibiotic class most frequently prescribed to treat UTIs in Western Europe and
North America is the fluoroquinolones. Fluoroquinolones are synthetic antibiotics derived
from nalidixic acid. Resistance to the synthetic fluoroquinolone antibiotics is increasing
among the organisms that cause UTIs.

Inhaltsverzeichnis (Table of Contents)

• I. INTRODUCTION AND GENERAL BACKGROUND
• 1. The bacterium Escherichia coli
• 2. Urinary tract infections (UTIs)
• 3. Resistance to antibiotics
• 3.1 Resistance mutations
• 3.2 Resistance to fluoroquinolones
• 3.2.1 Target site mutations
• 3.2.1.1 Mechanism of DNA gyrase and topoisomerase IV
• 3.2.2 Resistance caused by increased efflux
• 3.2.2.1 The mar-locus
• 3.3 Minimum inhibitory concentration
• 3.4 Mutation rate versus resistance
• 4. The system
• II. AIM OF THE STUDY
• III. MATERIAL AND METHODS
• 1. Growth media and solutions
• 1.1 Induction of the plasmid pKD20
• 1.2 Solution for Sequencing
• 2. Antibiotics
• 3. Bacterial strains and plasmids
• 3.1 plasmid pKD20
• 3.2 Plasmid pCP16
• 4. PCR general
• 4.1 PCR Beads
• 4.2 PCR for amplifying tetRA from Tn10
• 4.3 PCR for amplifying tetRA from the plasmid pCP16
• 4.4 Primers for PCR and DNA sequencing
• 5. Treatment with Dpn1
• 6. Agarose gel electrophoresis
• 7. Gel extraction
• 8. Measurement of DNA concentration
• 9. Using single-stranded oligonucleotides to introduce point mutations into a gene
• 10. Preparing electrocompetent cells with the λ Red system induced
• 10.1 Chromosomal λ-Red system
• 10.2 Plasmid-borne λ-Red system
• 11. Electroporation
• 12. Recombined chromosome
• 13. The use of Flp
• 14. Transformation with heat shock
• 15. Thermal cycle sequencing
• 15.1 Sequencing protocol
• 16. P1 phage preparation and transduction
• 17. The general scheme of this recombination method
• IV. RESULTS
• 1. Optimising electroporation efficiency
• 2. Recombining tetracycline resistance into muts
• 3. Recombining FRT-tetracycline resistance-FRT into muts and marR
• 4. Introducing a point mutation into the genes gyrA and parE
• 5. Removing tetracycline resistance using Flp
• 6. Assaying recombinant phenotypes
• 7. Sequencing recombination junctions
• V. DISCUSSION
• 1. General
• 2. Electroporation efficiency
• 3. Dpn1 treatment
• 4. Recombinants
• 5. Chromosome versus plasmid
• 6. Using oligonucleotides

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This research focuses on introducing specific mutations into the Escherichia coli chromosome using linear transformation. The study aims to understand the mechanisms of antibiotic resistance in E. coli, specifically those related to fluoroquinolone resistance. The work explores the role of target site mutations, increased efflux, and the mar-locus in conferring resistance.

• Antibiotic resistance in Escherichia coli
• Mechanisms of fluoroquinolone resistance
• Target site mutations
• Increased efflux and the mar-locus
• Linear transformation and genetic manipulation

Zusammenfassung der Kapitel (Chapter Summaries)

• I. INTRODUCTION AND GENERAL BACKGROUND: This section provides an overview of E. coli, urinary tract infections, and antibiotic resistance. It discusses the role of target site mutations, efflux pumps, and the mar-locus in resistance to fluoroquinolones. The chapter introduces the system used for introducing mutations into the E. coli chromosome, involving linear transformation and the λ Red recombination system.
• II. AIM OF THE STUDY: This chapter outlines the specific objectives of the research, which includes developing a method for introducing mutations into the E. coli chromosome and investigating the impact of these mutations on antibiotic resistance.
• III. MATERIAL AND METHODS: This chapter describes the materials and methods used in the study. It details the bacterial strains, plasmids, growth media, antibiotics, and techniques employed for DNA manipulation, including PCR, electroporation, and sequencing. The chapter also provides a detailed overview of the λ Red recombination system and its application for introducing mutations into the E. coli chromosome.
• IV. RESULTS: This section presents the results of the research. It describes the successful optimization of the electroporation technique for introducing mutations into the E. coli chromosome, demonstrating the efficient recombination of antibiotic resistance genes into specific regions of the genome. The chapter also includes data on the removal of antibiotic resistance genes using the Flp recombinase system and the characterization of recombinant phenotypes.
• V. DISCUSSION: This chapter discusses the significance of the research findings, analyzing the efficiency of the developed methods for introducing mutations and the implications of these mutations for antibiotic resistance in E. coli. The discussion includes insights into the role of different mechanisms of resistance and explores the advantages of using linear transformation and the λ Red recombination system for genetic manipulation in E. coli.

Schlüsselwörter (Keywords)

The study delves into the areas of antibiotic resistance, particularly fluoroquinolone resistance in E. coli, and explores the genetic mechanisms underlying this phenomenon. Key research focuses include target site mutations, increased efflux, the mar-locus, linear transformation, and the λ Red recombination system. These elements are essential for understanding the intricate pathways leading to antibiotic resistance and the potential development of strategies to combat it.

Details

• College: University of Innsbruck (Institute for Microbiology)
• Grade: 1 (A)
• Author: Peter Thaler (Author)
• Publication Year: 2003
• Pages: 48
• Catalog Number: V20071
• ISBN (eBook): 9783638240642
• Language: English
• Tags: Introducing Escherichia