1. Effect of Plasmid Concentration on Bacterial Transformation
Emmanuel J. Eppinger
Campus School of Carlow University
8th Grade

2. 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.

3. 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

4. What is Selection?
The use of an antibiotic resistance causing gene to isolate only transformed bacteria
gene of interest
Transformation
Competent Cells
Ampicillin Resistance Causing Gene
Ampicillin
Transformed Colony

5. Transformation Efficiency
Number of Transformed Colonies
Amount of Plasmid Added to Bacteria
Transformation Efficiency (TE)
TE =
# of

6. 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

7. 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.

8. Materials Experiment-Specific Supplies Disposable Lab Supplies
Goggles, gloves, ice, sterile distilled water
1 mL of competent DH5 alpha E. Coli cells
Sterile micro pipette tips
15 µL of Puc 18 ampicillin resistance causing plasmid at .82 µg of plasmid per µL of solution
Sterile1.5 mL micro tubes
Lab Equipment
6 mL of sterile LB nutrient agar
Timer
23 ampicillin positive agar plates
Closed test tube rack
1 ampicillin negative agar plates
37˚C Incubator
Sterile spreader bars
Turn table
Labeling utensil
Pipetter

9. Procedure Step 1: Prepare Plasmid
Wear goggles and gloves
Keep plasmid on ice for entire dilution
Create various concentrations of plasmid by diluting with water according to table
For each concentration, add 2µL of diluted plasmid to a 1.5 mL tube labeled with the concentration
Concentration
Plasmid (µL)
Water (µL) 1x 6
.5x 3
.1x 1 9
.05x 19
.01x 99
.001x
999
.0001x
9999

10. Procedure Step 2: Transform Bacteria
Add 100 µL of the competent cells to each of the labeled1.5mL tubes
Re-suspend the cells and plasmid by drawing in 30 µL of the solution and adding it back repeatedly for 5 to 10 seconds
Keep the tubes on ice for 40 minutes
Add 450 µL of LB nutrient agar solution to each tube
Place the tubes in pre-heated 37˚C water in a 37˚C incubator for 5 minutes to allow plasmids to be drawn into cells
Place the tubes back on ice

11. Procedure Step 3: Plate Bacteria
Re-suspend all the solutions in each of the tubes
For each concentration, add 100 µL of the solution to each of 3 ampicillin positive plates
Spread 100 µL of cells on both an ampicillin positive and negative plate (controls)
Spread the liquid using a spreader bar and a turn table
Invert the plates and incubate at 37˚C for 48 hours
Procedure Step 4: Analyze Results
Count and record the number of colonies on each plate
Compute the transformation efficiencies for each plate
Run an ANOVA test on the transformation efficiencies
Sterilize the plates

12. 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

13. Results: Number of Colonies at Each DNA Concentration
Trial 1
Trial 2
Trial 3
Average
Negative Control
.0001x
.001x 41 47 54
.01x
566
345
399
437
.05x
764
762
638
721
.1x
1200
916
1412
1176
.5x
1244
1905
1422
1524 1x
2448
2224
2044
2239
Positive Control
Lawn NA
Concentration of DNA

14. Results: Images Negative Control had no colonies.
0.01x had only about 400 colonies.
0.5x had about 1500 colonies.
Positive Control was a Lawn.

15. Results: Colonies for all Concentrations
Number of Colonies
Concentration of DNA (x)

16. Results: Transformation Efficiencies
Average Number of Colonies Transformed
Amount of DNA Added
(µg)
Transformation Efficiency
(Number of Transformed Colonies per µg of DNA)
.0001x
.001x 47
28,862
.01x
437
0.0164
26,626
.05x
721
0.082
8797
.1x
1176
0.164
7171
.5x
1524
0.82
1858 1x
2239
1.64
1365
Concentration of DNA

17. Results: Transformation Efficiencies
Number of Transformed Colonies per µg of DNA
Trend Line
Concentrations of DNA (x)

18. 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 different from each other?
SUMMARY
Groups
Count
Sum
Average
Variance 3
0.0164
0.082
774737
0.164
0.82
173977
1.64
15231
ANOVA
Source of Variation SS df MS F
P-value
F crit
Between Groups
2.64E+09 6
4.4E+08
2.32E-08
Within Groups
1.37E+08 14
Total
2.77E+09 20
P-value shows the probability that the results are due to chance
The ANOVA showed that the mean transformation efficiencies at each DNA concentration were significantly different from each other (p=2.32E-08).

19. 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 x which had 0 colonies per µg 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.
This point is between µg of DNA (0.0001x) and µg (0.001x) of DNA.

20. 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.
Statistical testing from the ANOVA supported these results since the p-value was 2.32E-08, showing the remote chance that all the data were random variations.

21. 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 covered by colonies rather than the number of colonies

22. 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

23. Works Cited
Maczulak, Anne. Allies and Enemies: How the World Depends on Bacteria. Upper Saddle River: Pearson Education, Inc., Plattsburg.edu. 10 November.< ansformation.html.> ScienceBuddies.org. 10 November.< -fair-projects/project_ideas/BioChem_p013.shtml.> Wikipedia.org. 12 October.< n_(genetics).> Wikipedia.org. 5 November 2012.<

24. Acknowledgements
Mrs. Wojociechowski Middle School Science Teacher, Campus School of Carlow University Mr. Krotec High School Biology Teacher, Central Catholic High School