Introducing specific mutations into the Escherichia coli chromosome using linear transformation

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 λ 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

7. Final conclusions

VI. SUMMARY

VII. REFERENCES

VIII. APPENDIX

1. Abbreviations

2. Codon table

Objectives and Topics

The primary goal of this thesis is to adapt and validate the linear transformation method (recombineering) for engineering specific mutations into the chromosome of Escherichia coli, particularly to investigate the relationship between genetic mutations and antibiotic resistance phenotypes. The research focuses on inactivating specific genes and attempting to introduce precise point mutations to understand clinical antibiotic resistance mechanisms.

• Application of the λ Red recombination system for chromosome engineering.
• Construction of gene inactivations in the mutS and marR genes using selectable markers.
• Use of Flp-recombinase to remove selectable markers after successful recombination.
• Investigation of antibiotic resistance phenotypes and mutation rates in engineered strains.
• Optimization of electroporation efficiency for genomic modification.

Excerpt from the Book

Summary of Chapters

I. INTRODUCTION AND GENERAL BACKGROUND: Provides fundamental information on E. coli, the clinical relevance of UTIs, and the mechanisms of antibiotic resistance, including target site alterations and efflux pumps.

II. AIM OF THE STUDY: Outlines the objective of using linear transformation to bridge the gap between genotype and phenotype regarding fluoroquinolone resistance.

III. MATERIAL AND METHODS: Details the experimental setup, including growth media, PCR protocols, the λ Red recombination system, and sequencing techniques.

IV. RESULTS: Presents the successful recombination of resistance markers into E. coli chromosomes and the evaluation of the resulting phenotypes.

V. DISCUSSION: Analyzes the efficiency of the recombination methods, the impact of Dpn1 treatment, and compares plasmid-borne versus chromosomal expression systems.

VI. SUMMARY: Concludes the thesis by summarizing the successful implementation of the recombineering technique and the insights gained into gene inactivation.

VII. REFERENCES: Lists the academic literature and sources cited throughout the thesis.

VIII. APPENDIX: Contains supporting technical information including a list of abbreviations and a codon table.

Keywords

Escherichia coli, antibiotic resistance, linear transformation, λ Red system, recombineering, mutS, marR, fluoroquinolones, gene inactivation, PCR, electroporation, Flp-recombinase, phenotype, mutation rate, DNA sequencing.

Frequently Asked Questions

What is the primary focus of this thesis?

The work focuses on utilizing the λ Red linear recombination system to engineer specific genomic mutations in Escherichia coli to better understand the link between genotype and antibiotic resistance.

What are the central themes of the research?

The research explores antibiotic resistance mechanisms, chromosome engineering techniques, and the functional validation of genetic mutations in a model organism.

What is the ultimate objective of the study?

The primary aim is to establish a reliable method for evaluating how specific chromosomal mutations contribute to the phenotypes of antibiotic-resistant bacteria.

Which scientific method is utilized?

The author employs "recombineering," a process using bacteriophage Lambda proteins (λ Red) to facilitate homologous recombination of linear PCR-generated DNA fragments into the bacterial chromosome.

What does the main body of the work cover?

The main body describes the optimization of electroporation, the construction of gene knock-outs in mutS and marR, the removal of markers via Flp-recombinase, and the assessment of resistance phenotypes.

Which keywords characterize this work?

Key terms include Escherichia coli, λ Red system, recombineering, antibiotic resistance, and gene inactivation.

How is the marR gene associated with resistance?

The thesis demonstrates that inactivating the marR repressor leads to the upregulation of the AcrAB efflux pump, resulting in increased resistance to multiple antibiotics including norfloxacin.

Why was the mutS gene chosen for study?

mutS encodes a mismatch repair protein; its inactivation was chosen to create a high-mutation-rate strain pair, enabling the measurement of the rate of resistance evolution in vivo.