Introducing specific mutations into the Escherichia coli chromosome using linear transformation E-Book

Peter Thaler

• Herausgeber: GRIN Verlag
• Sprache: Englisch

Veröffentlichungsjahr: 2003

Beschreibung

Diploma Thesis from the year 2003 in the subject Biology - Micro- and Molecular Biology, grade: 1 (A), University of Innsbruck (Institute for Microbiology), language: English, abstract: 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.

Leseprobe

Table of Content

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

3.2.2 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

3.2 Plasmid pCP16

4. PCR general

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. Final conclusions

VI. SUMMARY

VII. REFERENCES

VIII. APPENDIX

1. Abbrevations

2. Codon table

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ACKNOWLEDGEMENTS

The practical part of this study was made in the time from November 2002 until May 2003 in the Department of Cell and Molecularbiology, Uppsala University, Sweden. I want to thank very much Prof. Diarmaid Hughes for excellent supervising and sharing all the knowledge in this particular field of science.

I am also very grateful to Prof. Franz Allerberger, Institute of Social Medicine and Hygiene, University Innsbruck for excellent supervising and giving me the great chance to present my work at the antimicrobial resistance conference in Bethesda, USA. Great Thanks to Patricia Komp-Lindgreen and Linda Marcusson for the helpful advices and the fun time in the lab.

Thanks to Prof. Manfred P. Dierich for the help from the Institute of Social Medicine and Hygiene.

Thanks to Santanu Dasgupta for the Phage lysate and helpful advices. Thanks for all the support and the helpful discussions to the collaborators from the Department of Cell and Molecularbiology, Uppsala University.

Thanks to my parents Waltraud and Paul for taking care of me and alltime supporting my studies.

Thanks to my sister Michaela for the belief in me. Thanks to Ingo for the helpful discussions during writing. Thanks to all my friends which where beside me during my studies.

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7. Gel extraction 23

8. Measurement of DNA concentration 24

9. Using single-stranded oligonucleotides to introduce point mutations into a gene 24

10. Preparing electrocompetent cells with theλRed system induced 25

10.1 Chromosomalλ-Redsystem 25

10.2 Plasmid-borneλ-Redsystem 26

11. Electroporation 26

12. Recombined chromosome 27

13. The use of Flp 27

14. Transformation with heat shock 27

15. Thermal cycle sequencing 2815.1 Sequencing protocol 29

16. P1 phage preparation and transduction 30

17. The general scheme of this recombination method 30IV. RESULTS 32

1. Optimising electroporation efficiency 32

2. Recombining tetracycline resistance intomutS32

3. Recombining FRT-tetracycline resistance-FRT intomutSandmarR32

4. Introducing a point mutation into the genesgyrAandparE33

5. Removing tetracycline resistance using Flp 33

6. Assaying recombinant phenotypes 34

7. Sequencing recombination junctions 35V. DISCUSSION 37

1. General 37

2. Electroporation efficiency 38

3. Dpn1 treatment 38

4. Recombinants 39

5. Chromosome versus plasmid 39

6. Using oligonucleotides 40

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I. INTRODUCTION AND GENERAL BACKGROUND

1. The bacteriumEscherichia coli

In 1885 Theodor Escherich described the gram negative bacteriumEscherichia 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. colican also cause diseases like diarrhea, inflammation of the urinary tract or the gall bladder.

Figure 1: Electron microscope picture of the bacteriaE. coli(from http://commtechlab.msu .edu/sites/dlc-me/zoo/Pf07002.jpg; accessed 11.07.03).

2. Urinary tract infections (UTIs)

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 (Canbazet 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 isE. coli(60-80 %), usually originating from the patients own faecal flora, followed byStaphylococcus saprophyticus(10 %),Klebsiella sp.,other Gram negative bacteria and enterococci (Burman and Olsson-Liljequist,