Effect of Plasmid Concentration on Bacterial Transformation
Emmanuel J. EppingerCampus School of Carlow University8th Grade
What is Transformation?
PlasmidA circular piece of DNA
Competent CellA cell that is able to
accept DNA
Transformed CellA cell that has new DNA
Transformation was discovered in 1928 by Fredrick Griffith.
The addition of foreign deoxyribonucleic acid (DNA) into a bacterium
Uses of Bacterial Transformation
To mass produce proteinse.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?
Transformation
Ampicilli
n
Ampicillin Resistance Causing Gene
Transformed Colony
The use of an antibiotic resistance causing gene to isolate only transformed bacteriagene of interest
Competent Cells
Transformation Efficiency
Transformation Efficiency (TE)
Number of Transformed Colonies
Amount of Plasmid Added to Bacteria
TE =
# of
PurposeDuring 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
HypothesisIf
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.
MaterialsExperiment-Specific
Supplies◦1 mL of competent DH5 alpha E. Coli cells◦15 µL of Puc 18 ampicillin resistance causing plasmid at .82 µg of plasmid per µL of solution◦6 mL of sterile LB nutrient agar◦23 ampicillin positive agar plates◦1 ampicillin negative agar plates
Disposable Lab Supplies◦ Goggles, gloves, ice, sterile
distilled water◦ Sterile micro pipette tips◦ Sterile1.5 mL micro tubes
Lab Equipment◦ Timer◦ Closed test tube rack◦ 37˚C Incubator◦ Sterile spreader bars◦ Turn table◦ Labeling utensil◦ Pipetter
Procedure Step 1: Prepare Plasmido Wear goggles and gloves
o Keep plasmid on ice for entire dilution1. Create various concentrations of plasmid by
diluting with water according to table
2. 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 0
.5x 3 3
.1x 1 9
.05x 1 19
.01x 1 99
.001x 1 999
.0001x 1 9999
Procedure Step 2: Transform Bacteria
3. Add 100 µL of the competent cells to each of the labeled1.5mL tubes
4. Re-suspend the cells and plasmid by drawing in 30 µL of the solution and adding it back repeatedly for 5 to 10 seconds
5. Keep the tubes on ice for 40 minutes6. Add 450 µL of LB nutrient agar solution to
each tube7. 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
8. Place the tubes back on ice
Procedure Step 3: Plate Bacteria9. Re-suspend all the solutions in each of the tubes10. For each concentration, add 100 µL of the solution
to each of 3 ampicillin positive plates11. Spread 100 µL of cells on both an ampicillin
positive and negative plate (controls)12. Spread the liquid using a spreader bar and a turn
table13. Invert the plates and incubate at 37˚C for 48 hours
Procedure Step 4: Analyze Results14. Count and record the number of colonies on each plate15. Compute the transformation efficiencies for each plate16. Run an ANOVA test on the transformation efficiencies17. Sterilize the plates
VariablesDependent 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
ControlsPositive Control: Untransformed bacteria on ampicillin negative plateNegative Control: Untransformed bacteria on ampicillin positive plate
Results: Number of Colonies at Each DNA Concentration
Trial 1 Trial 2 Trial 3 AverageNegative Control 0 0.0001x 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 1244 1905 1422 1524
1x 2448 2224 2044 2239Positive Control Lawn NA
Conce
ntr
ati
on o
f D
NA
Results: Images
Positive Control was a Lawn.
0.5x had about 1500 colonies.
Negative Control had no colonies.
0.01x had only about 400 colonies.
Results: Colonies for all Concentrations
Num
ber
of
Colo
nie
s
Concentration of DNA (x)
Negiti
ve C
ontro
l
0.00
01x
0.00
1x
0.01
x
0.05
x0.
1x0.
5x 1x0
500
1000
1500
2000
2500
Average Number of Transformed Colonies
Average Number of Transformed ColoniesPolynomial (Av-erage Number of Transformed Colonies)
Trend Line
Results: Transformation Efficiencies
Average Number of
Colonies Transformed
Amount of DNA Added
(µg)
Transformation Efficiency(Number of
Transformed Colonies per µg of DNA)
.0001x 0 0.000164 0
.001x 47 0.00164 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
Conce
ntr
ati
on o
f D
NA
Results: Transformation EfficienciesN
um
ber
of
Transf
orm
ed C
olo
nie
s per
µg
of
DN
A
Concentrations of DNA (x)
0.00
01x
0.00
1x
0.01
x
0.05
x0.
1x0.
5x 1x0
5000
10000
15000
20000
25000
30000
35000
Number of Transformed Colonies per µg of DNA
Number of Trans-formed Colonies per µg of DNAPolynomial (Num-ber of Transformed Colonies per µg of DNA)
Trend Line
Results Table 3: ANOVA Test
SUMMARYGroups Count Sum Average Variance
0.000164 3 0 0 0
0.00164 3 86585.37 28861.79 157396390.0164 3 79878.05 26626.02 49354303
0.082 3 26390.24 8796.75 7747370.164 3 21512.2 7170.73 2302796
0.82 3 5574.39 1858.13 1739771.64 3 4095.122 1365.04 15231
ANOVASource of Variation SS df MS F P-value F crit
Between Groups 2.64E+09 6 4.4E+08 45.02287 2.32E-08 2.847726Within Groups 1.37E+08 14 9765812
Total 2.77E+09 20
The ANOVA showed that the mean transformation efficiencies at each DNA concentration were significantly different from each other (p=2.32E-08).
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?
P-value shows the probability that the results are due to chance
ConclusionThe 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 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 0.000164 µg of DNA (0.0001x) and 0.00164 µg (0.001x) of DNA.
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.
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
Future ResearchImproving 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 CitedMaczulak, 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.<http://en.wikipedia.org/wiki/Transformato
n_(genetics).>
Wikipedia.org. 5 November 2012.<http://en.wikipedia.org/wiki/DNA.>
Acknowledgements
Mrs. WojociechowskiMiddle School Science
Teacher, Campus School of Carlow University
Mr. KrotecHigh School Biology Teacher,Central Catholic High School