Transcript Doyle pH and transformation PJAS2011

By: Colleen Doyle
Oakland Catholic High School
Does
pH have an
effect on the
transformation of E.
coli?
 Are
human influences on
the environment affecting
the behavior of prokaryotic
organisms?
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The measure of acidity or basicity of an aqueous
solution
pH is measured on a scale of 0-14
The lower the pH of a solution the more acidic it is, the
higher the pH the more basic it is
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Worldwide problem
Caused by pollution of
sulfur dioxide and
nitrogen oxide
Often originates from
smokestacks, vehicle
exhaust, and burning
fossil fuels
Average pH of 5.6
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Gram negative, rod shaped bacterium, commonly
found in the environment and the intestines of
warm blooded animals
One of the most studied prokaryotic organisms in
microbiology
Although most strains of E. coli are harmless some
types can be harmful and can cause food poisoning
in humans.
Used as a model for prokaryotes in this experiment
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DH5-alpha was used in the
experiment. It is not resistant to
ampicillin; commonly used host
for ampr.
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Cells that absorb extraneous DNA
and express a new characteristic
are referred to as transformed.
Recombinant DNA technology
often makes use of naturally
occurring vectors of DNA.
Plasmids commonly used to
transform cells.
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A plasmid is extraneous DNA that is not
chromosomal.
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Often employed as a vector in biotechnology to
carry new genes into a host cell.
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pGEM 7 is a much utilized plasmid containing a
resistance gene to ampicillin.
AMP r
Null:
 Varying pH will have no effect on the
transformation of E. coli.
Alternative:
 pH variation will alter transformation
efficiency in DH5-alpha E. coli.
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Micropipettes + Sterile Tips
Micro tubes
Spreader Bars
Ethanol and Bunsen burner
Incubator
pH indicator strips
Acids/Bases
– HCl (acid)
– NH3 (base)
Sterile Dilution Fluid (100mM KH2PO4, 100mM K2HPO4, 10mM
MgSO4, 1mM NaCl)
Sterile water
LB agar plates (1% Tryptone, 0.5% Yeast Extract, 1% NaCl)
LB-amp agar plates
DH5-Alpha E. coli
pGEM 7 Plasmid DNA
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10µl aliquots of sterile acidic or basic solution was
added to SDF to create the following pH’s: 1,3,
5,7, 8,10 (standardizing volumes)
200µl of DH-5 Alpha E. coli was added to each
sample
Each sample was incubated in ice for 45 minutes
They were heat shocked at 37° Celsius
500µl of each pH solution was administered on a
LB-amp agar plates and spread
Plates were incubated over night
Colonies on each plate were counted
Cell Survivorship
T-Critical Value: 3.62
Alpha Value: 0.05
pH
T-Value
Interpretation
1
1.11
Insignificant
3
3.34
Insignificant
5
8.23
Significant
8
0.72
Insignificant
10
8.77
Significant
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Calcium-competent DH5-Alpha E. coli tubes were thawed
and pooled to serve as a common cell stock
180µl of cells were added to six micro tubes
Aliquots of sterile acidic or basic solution was added (and
sterile distilled water to standardize volume) until the
desired pH was reached.
10µl of p-GEM 7 plasmid DNA was administered into each
solution
Each sample was incubated in ice for 45 minutes
Each sample was heat shocked at 37° Celsius for 5 minutes
50µl of each pH solution was distributed on LB-amp agar
plates and spread
Plates were incubated overnight
Colonies on each plate were counted
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Positive Control: Competent cells
administered on LB-agar plates grew a
lawn of bacteria
Negative Control: Competent cells, without
plasmid, administered on LB-amp plates
grew NO bacteria
Transformants
T-Critical Value: 3.92
Alpha Value: 0.05
pH
T-Value
Interpretation
1
26.92
Significant
3
26.38
Significant
5
0.27
Insignificant
8
8.38
Significant
10
26.83
Significant
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The Dunnett’s test shows that the majority of
each of the individual results varied
significantly from the control.
pH alters the efficiency of transformation in
DH5-alpha E. coli.
My Alternative Hypothesis is supported
My Null Hypothesis is rejected for the pHs of
1, 3, 8, 10
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Were competent
cells homogeneous
in concentration?
Only one plasmid
utilized (various
sizes
recommended)
NH3 might have a
toxic affect
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Test different
prokaryotic
organisms
Test with different
strain of E. coli
Test with different
types of plasmids
Larger sample size
Test gene
expression
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AP Biology Class Test Edition
http://www.eoearth.org/article/PH
http://www.elmhurst.edu/~chm/vchembook/184ph.html
http://bcs.whfreeman.com/thelifewire8e/
http://www.epa.gov/acidrain/
http://faculty.plattsburgh.edu/donald.slish/Transformation.html
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X-gal substrate is used to indicate the
presence of an intact Lac Z. If Lac Z is intact,
β-galactosidase activity is restored, with
resulting cleavage of X-gal which leads to
characteristic blue colony phenotype.
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White colonies = AMPr, LAC Z disrupted
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Blue Colonies = AMPr and LAC Z intact
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The smaller the plasmid the easier it is to
enter cell membrane
pGEM 7 plasmid has an approximate size of
3.0 kb or kilobases (1kb=1,000 base pairs)
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Cell wall is thin, extra layer of lipopolysaccharide which adds
extra level of protection.
If the toxin enters the circulatory system it causes a toxic
reaction.
This outer membrane protects the bacteria from several
antibiotics.
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All solutions are acidic or basic, this depends on
their concentration of H+ in Moles per liter
(Molarity)
Logarithm of H+ is log10 of 100
Ex: Pure water has a H+ concentration of 10-7 M,
its pH equals log10 (10-7)=-(-7) or 7 which is the
pH of pure water