Transcript Eppinger PJAS Slides 2013x

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
Competent Cell
A circular piece of DNA
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
Transformation
Ampicillin
Resistance
Causing Gene
Competent Cells
Transformed Colony
Transformation Efficiency
Number of
Transformed
Colonies
Transformation
Efficiency (TE)
Amount of
Plasmid Added
to Bacteria
#
of
TE =
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

◦ Goggles, gloves, ice, sterile distilled
water
◦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
◦ 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 Plasmid
o
o
1.
2.
Wear goggles and gloves
Keep plasmid on ice for entire dilution
Create various concentrations of plasmid by diluting
with water according to table
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
For each concentration, add 2µL 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 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
Procedure Step 3: Plate Bacteria
Re-suspend all the solutions in each of the tubes
10. For each concentration, add 100 µL of the solution to
each of 3 ampicillin positive plates
11. 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 table
13. Invert the plates and incubate at 37˚C for 48 hours
9.
Procedure Step 4: Analyze Results
Count and record the number of colonies on each plate
15. Compute the transformation efficiencies for each plate
16. Run an ANOVA test on the transformation efficiencies
17. Sterilize the plates
14.
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 colonies.
0.01x had only
about 400 colonies.
Positive Control
was a Lawn.
Results: Colonies for all Concentrations
Average Number of Transformed Colonies
2500
2000
Number of Colonies
1500
Average Number of
Transformed Colonies
1000
Trend(Average
Line
Poly.
Number of
Transformed Colonies)
500
0
Concentration of DNA (x)
Concentration of DNA
Results: Transformation Efficiencies
Transformation
Efficiency
Average Number
of Colonies
Transformed
Amount of
DNA Added
(µg)
(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
Results: Transformation Efficiencies
Number of Transformed Colonies per µg of DNA
Number of Transformed Colonies per µg of
DNA
35000
30000
25000
Number of
Transformed Colonies
per µg of DNA
Trend(Number
Line
Poly.
of
Transformed Colonies
per µg of DNA)
20000
15000
10000
5000
0
Concentrations of DNA (x)
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
SUMMARY
from each other?
Groups
Count
Sum
Average
Variance
0.000164
3
0
0
0
0.00164
0.0164
0.082
0.164
0.82
1.64
3
3
3
3
3
3
86585.37
79878.05
26390.24
21512.2
5574.39
4095.122
28861.79
26626.02
8796.75
7170.73
1858.13
1365.04
15739639
49354303
774737
2302796
173977
15231
ANOVA
Source of
Variation
Between
Groups
2.64E+09
6
4.4E+08
Within Groups
1.37E+08
14
9765812
Total
2.77E+09
20
SS
df
MS
F
45.02287
P-value shows the
probability that the results
are due to chance
P-value
2.32E-08
F crit
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 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 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.<http://en.wikipedia.org/wiki/Transformato
n_(genetics).>
Wikipedia.org. 5 November
2012.<http://en.wikipedia.org/wiki/DNA.>
Acknowledgements
Mrs. Wojociechowski
Middle School Science Teacher,
Campus School of Carlow University
Mr. Krotec
High School Biology Teacher,
Central Catholic High School