pH Effects on the Transformation of Escherichia coli

1. pH Effects on the Transformation of Escherichia coli By: Colleen Doyle Oakland Catholic High School

2. Problem • Does pH have an effect on the transformation of E. coli?

3. Are human influences on the environment affecting the behavior of prokaryotic organisms?

4. pH • The measure of acidity or basicity of an aqueous solution • pH is measured on a scale of 0-14 • The lower the pH of a solution the more acidic it is, the higher the pH the more basic it is

5. Acid Rain • Worldwide problem • Caused by pollution of sulfur dioxide and nitrogen oxide • Often originates from smokestacks, vehicle exhaust, and burning fossil fuels • Average pH of 5.6

6. Escherichia coli (E. coli) • Gram negative, rod shaped bacterium, commonly found in the environment and the intestines of warm blooded animals • One of the most studied prokaryotic organisms in microbiology • Although most strains of E. coli are harmless some types can be harmful and can cause food poisoning in humans. • Used as a model for prokaryotes in this experiment

7. DH5-Alpha E. coli • DH5-alpha was used in the experiment. It is not resistant to ampicillin; commonly used host for ampr.

8. Transformation • Cells that absorb extraneous DNA and express a new characteristic are referred to as transformed. • Recombinant DNA technology often makes use of naturally occurring vectors of DNA. • Plasmids commonly used to transform cells.

9. pGEM 7 Plasmid DNA • A plasmid is extraneous DNA that is not chromosomal. • Often employed as a vector in biotechnology to carry new genes into a host cell. • pGEM 7 is a much utilized plasmid containing a resistance gene to ampicillin. AMP r

10. Hypotheses Null: • Varying pH will have no effect on the transformation of E. coli. Alternative: • pH variation will alter transformation efficiency in DH5-alpha E. coli.

11. Materials • Micropipettes + Sterile Tips • Micro tubes • Spreader Bars • Ethanol and Bunsen burner • Incubator • pH indicator strips • Acids/Bases • HCl (acid) • NH3(base) • Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM MgSO4, 1mM NaCl) • Sterile water • LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl) • LB-amp agar plates • DH5-Alpha E. coli • pGEM 7 Plasmid DNA

12. Cell Survivorship Procedure • 10µl aliquots of sterile acidic or basic solution was added to SDF to create the following pH’s: 1,3, 5,7, 8,10 (standardizing volumes) • 200µl of DH-5 Alpha E. coli was added to each sample • Each sample was incubated in ice for 45 minutes • They were heat shocked at 37° Celsius • 500µl of each pH solution was administered on a LB-amp agar plates and spread • Plates were incubated over night • Colonies on each plate were counted

13. Samples: 1 3 5 7 8 10

14. Example: pH 10

16. Significant Variation (Dunnett's Test) Cell Survivorship T-Critical Value: 3.62 Alpha Value: 0.05

17. Transformation Procedure • Calcium-competent DH5-Alpha E. coli tubes were thawed and pooled to serve as a common cell stock • 180µl of cells were added to six micro tubes • Aliquots of sterile acidic or basic solution was added (and sterile distilled water to standardize volume) until the desired pH was reached. • 10µl of p-GEM 7 plasmid DNA was administered into each solution • Each sample was incubated in ice for 45 minutes • Each sample was heat shocked at 37° Celsius for 5 minutes • 50µl of each pH solution was distributed on LB-amp agar plates and spread • Plates were incubated overnight • Colonies on each plate were counted

18. Controls • Positive Control: Competent cells administered on LB-agar plates grew a lawn of bacteria • Negative Control: Competent cells, without plasmid, administered on LB-amp plates grew NO bacteria

20. Significant Variation (Dunnett's Test) Transformants T-Critical Value: 3.92 Alpha Value: 0.05

21. Conclusion • The Dunnett’s test shows that the majority of each of the individual results varied significantly from the control. • pH alters the efficiency of transformation in DH5-alpha E. coli. • My Alternative Hypothesis is supported • My Null Hypothesis is rejected for the pHs of 1, 3, 8, 10

22. Limitations Extensions • Test different prokaryotic organisms • Test with different strain of E. coli • Test with different types of plasmids • Larger sample size • Test gene expression • Were competent cells homogeneous in concentration? • Only one plasmid utilized (various sizes recommended) • NH3 might have a toxic affect

23. Sources 1. AP Biology Class Test Edition 2. http://www.eoearth.org/article/PH 3. http://www.elmhurst.edu/~chm/vchembook/184ph.html 4. http://bcs.whfreeman.com/thelifewire8e/ 5. http://www.epa.gov/acidrain/ 6. http://faculty.plattsburgh.edu/donald.slish/Transformation.html

24. Dunnett’s Test

25. Gene Expression • X-gal substrate is used to indicate the presence of an intact Lac Z. If Lac Z is intact, β-galactosidase activity is restored, with resulting cleavage of X-gal which leads to characteristic blue colony phenotype. • White colonies = AMPr, LAC Z disrupted • Blue Colonies = AMPr and LAC Z intact

26. Plasmid Size • The smaller the plasmid the easier it is to enter cell membrane • pGEM 7 plasmid has an approximate size of 3.0 kb or kilobases (1kb=1,000 base pairs)

27. Gram Negative Bacterium • Cell wall is thin, extra layer of lipopolysaccharide which adds extra level of protection. • If the toxin enters the circulatory system it causes a toxic reaction. • This outer membrane protects the bacteria from several antibiotics.

28. The Chemistry of pH • All solutions are acidic or basic, this depends on their concentration of H+ in Moles per liter (Molarity) • Logarithm of H+ is log10 of100 • Ex: Pure water has a H+ concentration of 10-7 M, its pH equals log10 (10-7)=-(-7) or 7 which is the pH of pure water