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