Effect of Plasmid Concentration on Bacterial Transformation Emmanuel J. Eppinger Campus School of Carlow University 8th Grade What is Transformation? The addition of foreign deoxyribonucleic acid (DNA) into a bacterium Plasmid A circular piece of DNA Competent Cell A cell that is able to accept DNA Transformed Cell A cell that has new DNA Transformation was discovered in 1928 by Fredrick Griffith. Uses of Bacterial Transformation To mass produce proteins e.g. DRACO protein gives
immunity to all viruses (e.g., cold, flu, Ebola) To transfer traits among species e.g. Transfer of luminescence genes can be used to create secret codes To mass produce DNA DNA is multiplied each time a cell reproduces What is Selection? The use of an antibiotic resistance causing gene to isolate only transformed bacteria gene of interest Transformati on Ampicillin Resistance Causing Gene Competent Cells Transformed Colony illi
c i p m A n Transformation Efficiency Number of Transforme d Colonies Transformatio n Efficiency (TE) # of TE = Amount of Plasmid Added to Bacteria Purpose During transformation, not all bacteria are transformed.
Only some bacteria accept the plasmid. The purpose of this experiment is to determine whether the concentration of plasmid will affect the efficiency with which bacteria transform Hypothesis If the rate at which bacteria colonies transform is not proportional to the plasmid concentration, then the transformation efficiencies of different concentrations of plasmid will not be equal because transformation efficiency is a function of plasmid concentration. Materials Experiment- Specific Supplies 1 mL of competent DH5 alpha E. Coli cells 15 L of Puc 18 ampicillin resistance causing plasmid
at .82 g of plasmid per L of solution 6 mL of sterile LB nutrient agar 23 ampicillin positive agar plates 1 ampicillin negative agar plates Sterile micro pipette tips Sterile1.5 mL micro tubes Lab Equipment Timer Closed test tube rack 37C Incubator Sterile spreader bars Turn table Labeling utensil Pipetter
Procedure Step 1: Prepare Plasmid o Wear goggles and gloves o Keep plasmid on ice for entire dilution 1. Create various concentrations of plasmid by diluting with water according to table 2. Concentration Plasmid (L) Water (L) 1x 6 0 .5x 3 3
.1x 1 9 .05x 1 19 .01x 1 99 .001x 1 999 .0001x 1 9999
For each concentration, add 2L of diluted plasmid to a 1.5 mL tube labeled with the concentration Procedure Step 2: Transform Bacteria 3. 4. 5. 6. 7. 8. Add 100 L of the competent cells to each of the labeled1.5mL tubes Re-suspend the cells and plasmid by drawing in 30 L of the solution and adding it back repeatedly for 5 to 10 seconds Keep the tubes on ice for 40 minutes Add 450 L of LB nutrient agar solution to each tube Place the tubes in pre-heated 37C water in a 37C incubator for 5 minutes to allow plasmids to be drawn into cells Place the tubes back on ice Procedure Step 3: Plate Bacteria Re-suspend all the solutions in each of the tubes
10. For each concentration, add 100 L of the solution to each of 3 ampicillin positive plates 11. Spread 100 L of cells on both an ampicillin positive and negative plate (controls) 12. Spread the liquid using a spreader bar and a turn table 13. Invert the plates and incubate at 37C for 48 hours 9. Procedure Step 4: Analyze Results 14. Count and record the number of colonies on each plate 15. 16. 17. Compute the transformation efficiencies for each plate Run an ANOVA test on the transformation efficiencies Sterilize the plates Variables Dependent Variable Bacterial Transformation Efficiency: Number of colonies per plate/amount of DNA plated Independent Variable Concentration of Plasmid: 1x, .5x, .1x, .05x, .01x, .001x, and .0001x Controls Positive Control: Untransformed bacteria
on ampicillin negative plate Negative Control: Untransformed bacteria on ampicillin positive plate Results: Number of Colonies at Each DNA Concentration Trial 1 Concentration of DNA Negative Control Trial 2 Trial 3 Average .0001x 0 0 0 0 0 0 .001x 41
47 54 47 .01x 566 345 399 437 .05x 764 762 638 721 .1x 1200
916 1412 1176 .5x 1x 1244 1905 1422 1524 2448 2224 2044 2239 Positive Control Lawn
NA Results: Images Negative Control had no colonies. 0.5x had about 1500 0.01x had only about 400 colonies. Positive Control was Results: Colonies for all Concentrations Average Number of Transformed Colonies 2500 2000 1500 1000 Number of Colonies 500
0 e N l x x x x x x x o r 01 01 01 05 .1 .5 1 t 0 0 . . on .00 0.0 0 0 C 0 e tiv i Concentration of DNA (x) g Average Number of Transformed Colonies Polynomial (Average Trend LineNumber of Transformed Colonies) Results: Transformation
Concentration of DNA Efficiencies .0001x .001x .01x .05x .1x .5x 1x Average Number of Colonies Transformed 0 47 437 721 1176 1524 2239 Amount of DNA Added (g) 0.000164 0.00164
0.0164 0.082 0.164 0.82 1.64 Transformation Efficiency (Number of Transformed Colonies per g of DNA) 0 28,862 26,626 8797 7171 1858 1365 Results: Transformation Efficiencies Number of Transformed Colonies per g of DNA Number of Transformed Colonies per g of DNA 35000 30000 25000
20000 15000 10000 5000 0 Concentrations of DNA (x) Number of Transformed Colonies per g of DNA Polynomial (Number of Trend Line Transformed Colonies per g of DNA) Results Table 3: ANOVA Test ANOVA tests whether the mean transformation efficiencies at each concentration are really different from each other or whether the differences are only due to random variation. Are the averages significantly SUMMARY different from Groups Count Sum
Average Variance 0.000164 3 0 0 0 each other? 0.00164 3 86585.37 28861.79 15739639 0.0164 0.082 0.164 0.82 1.64 ANOVA Source of Variation Between Groups Within Groups Total 3 3 3
3 3 SS 79878.05 26390.24 21512.2 5574.39 4095.122 df 26626.02 8796.75 7170.73 1858.13 1365.04 MS 2.64E+09 6 4.4E+08 1.37E+08 14
9765812 2.77E+09 20 49354303 774737 2302796 173977 15231 F 45.02287 P-value shows the probability that the results are due to chance P-value F crit 2.32E-08 2.847726 The ANOVA showed that the mean transformation efficiencies at each DNA concentration were significantly different from
each other (p=2.32E-08). Conclusion The greater the DNA concentration, the greater the average number of transformed colonies. The transformation efficiencies were the opposite of the number of colonies: the lower the concentration of DNA, the higher the transformation efficiency. The exception was 0.0001x which had 0 colonies per g of DNA added. This suggests that Using lower concentrations of DNA is more efficient but that a point exists where so little plasmid is added that there is not enough to support a transformed colony. Therefore The original hypothesis, If the rate at which bacteria colonies transform is not proportional to the plasmid concentration, then the transformation efficiencies of
different concentrations of plasmid will not be equal because transformation efficiency is a function of plasmid concentration, was supported. The number of colonies for each concentration was not proportional to the concentration of the plasmid. Since they were not proportional, the transformation efficiencies were different and not equal. Changes to Improve Experiment Incubate for less time to prevent the formation of satellite colonies or multiple colonies merging Test with larger samples of bacteria and DNA Use an automated colony counter Measure the surface area Future Research Improving Transformation Efficiency
Test lower and higher concentrations of plasmid Test different types and sizes of plasmids Test different bacteria species e.g., HB101 Vary conditions of experiment e.g. specifications of heat shock Applications to other species Test transformation in plants, yeast, and animal cells Investigate potential uses in gene therapy in people Works Cited Maczulak, Anne. Allies and Enemies: How the World Depends on Bacteria. Upper Saddle River: Pearson Education, Inc., 2011. Plattsburg.edu. 10 November.< http://faculty.plattsburgh.edu/donald.slish/tr ansformation.html.> ScienceBuddies.org. 10 November.< http://www.sciencebuddies.org/science -fair-projects/project_ideas/ BioChem_p013.shtml.> Wikipedia.org. 12 October.
n_(genetics).> Wikipedia.org. 5 November Acknowledgements Mrs. Wojociechowski Middle School Science Teacher, Campus School of Carlow University Mr. Krotec High School Biology Teacher, Central Catholic High School
