ib biology ee on bacteria transformation by heat shock method using plasmid
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IB Biology EE on Bacteria Transformation by heat shock method using plasmid. Please cite and give proper reference to Seo Young if you use her workTRANSCRIPT
International Baccalaureate Diploma Program Extended Essay
Biology Determining the effect of changing the atomic size of divalent cations on
transformation efficiency of the heat-shock protocol on Escherichia Coli DH5 strain
Candidate Name: Seo Young Myaeng
Candidate Number: 002213-064
Word Count: 3394
Seo Young Myaeng Candidate Number: 002213-064
pg. 1/36
Acknowledgements:
I would like to acknowledge the following people for their support for making the investigation possible:
Mr. Lawrence Kok
Mr. Bob Johnson
Jason Ji Ho Rhim
Michael Seung-Hoon Shin
Kelly Chaehyun Lee
and
Taejon Christian International School
Seo Young Myaeng Candidate Number: 002213-064
pg. 2/36
Abstract
Transformation is a method used to change the genetic information of bacteria using a
circular DNA called plasmid. Plasmid cannot normally enter the bacteria because both the
plasmid and the bacteria cell membrane are negatively charged, and hence repel each other.
However, by adding divalent cations to the bacteria, the cell membrane becomes neutralized
and permeable to the plasmid. Because of divalent cations’ essential role in transformation,
the question of whether the type of cations used affects transformation efficiency was raised.
Therefore, this investigation will focus on the research equation: To what extent does
changing the atomic size of divalent cations affect transformation efficiency of the heat-
shock protocol on Escherichia Coli DH5 strain?
In the investigation, a heat-shock protocol on Eshcerichia Coli DH5 using pUC18 plasmid
will be performed. During the process, divalent cations of different sizes – Ca2+
, Mg2+
, and
Mn2+
– will be added in forms of chlorides. The pUC18 plasmid will make the bacteria
resistant to Ampicillin, a type of antibiotic. Thus, after transformation, the bacteria will be
plated on LB-Ampicillin plate, an environment full of the antibiotic, to see whether
transformation was successful. The plates will be incubated for 48 hours at 37°C before their
transformation efficiencies are calculated and compared.
The result was that though cations of different sizes were used in different transformation
processes, all transformations were successful. The values in different transformation
efficiencies were not the same, but according to the Tukey’s HSD Test, not much significant
difference was found between results of certain cations, such as Ca2+
and Mn2+
. From this, it
can be reflected that as long as divalent cations are used, transformation will be successful.
In conclusion, despite the change in atomic size of cations, as long as the cations are
divalent, transformation will be successful.
(294 words)
Seo Young Myaeng Candidate Number: 002213-064
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Table of Contents
Abstract ................................................................................................................................. 2
1.0 Introduction .................................................................................................................... 4
1.1 Rationale of the Study ............................................................................................ 4
1.2 DH5 Bacteria ....................................................................................................... 5
1.3 pUC18 Plasmid ...................................................................................................... 6
1.4 History of Transformation ...................................................................................... 7
2.0 Hypothesis ....................................................................................................................... 8
3.0 Variables ......................................................................................................................... 9
3.1 Manipulated Variable ............................................................................................. 9
3.2 Responding Variable .............................................................................................. 9
3.3 Constant Variable ................................................................................................. 10
4.0 Materials and Methods ................................................................................................ 11
4.1 Overall Methodology ........................................................................................... 11
4.2 Preparation of LB and LB-Ampicillin Plates ....................................................... 12
4.3 Preparation of Salt Solutions ................................................................................ 12
4.4 Culturing Bacteria Stock ...................................................................................... 12
4.5 Transformation ..................................................................................................... 13
5.0 Data Collection ............................................................................................................. 14
5.1 Raw Data .............................................................................................................. 14
5.1-1 Qualitative Data ................................................................................... 14
5.1-2 Quantitative Data ................................................................................. 16
5.2 Calculation of Transformation Efficiency............................................................ 17
5.3 Data Analysis ....................................................................................................... 18
6.0 Statistical Analysis........................................................................................................ 20
6.1 ANOVA Test........................................................................................................ 20
6.2 Tukey’s HSD Test ................................................................................................ 21
7.0 Evaluation ..................................................................................................................... 22
7.1 Explanation........................................................................................................... 22
7.2 Uncertainties and Limitations .............................................................................. 23
7.3 Ways to Improve .................................................................................................. 25
7.4 Further Investigation ............................................................................................ 26
8.0 Conclusion ..................................................................................................................... 27
9.0 Appendix ....................................................................................................................... 28
10.0 References ................................................................................................................... 36
Seo Young Myaeng Candidate Number: 002213-064
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1.0 Introduction
1.1 Rationale of the Study
During transformation, bacteria cannot take in plasmid by itself due to repulsion. Therefore,
the heat-shock method is used to make the bacteria more permeable to plasmid. First, divalent
cations1 are added in forms of chloride solutions. The standard chloride used during
transformation is calcium chloride or CaCl2. The solution will then dissociate into Ca2+
and
Cl- ions. Ca
2+ will then neutralize the negative charge on cell wall and membrane so that the
plasmid will no longer be repelled, and thus enter the bacteria easily.
Figure 1: Role of CaCl2 in transformation
[1]
However, just because Ca2+
is commonly used, it does not mean it is not the only type of
cations that may be used. There may be situations where there is a lack of CaCl2. Then it will
be necessary to know whether other divalent cations can be used to perform transformation.
Through this investigation, I will use cations of the same charge (2+) but of different atomic
sizes to observe whether it is possible to transform bacteria using solution other than CaCl2.
Therefore my research question will be: To what extent does changing the atomic size of
divalent cations affect transformation efficiency of the heat-shock protocol on
Escherichia Coli DH5 strain?
1 Molecules with the charge of 2+
Seo Young Myaeng Candidate Number: 002213-064
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1.2 DH5 Bacteria
Figure 2: Magnified image of DH5 [2]
DH5 [3] is a type of Escherichia Coli2 bacteria that will be used in the investigation. The
general E. Coli bacteria are found in the lower intestines of endotherms3, and rarely cause
harm other than through food poisoning. The bacteria can even be favorable to humans[4]
since they produce vitamin K and are used in medicines for gastroenterological diseases4.
Other than these general traits of E. Coli bacteria, the DH5 have their own specific
characters that are important for this investigation. First, they have relatively thin
peptidoglycan layer5 and can allow plasmid to enter the cell more easily. This will help
lessen the possibility of error from unsuccessful transformation. Also, DH5 can uptake
large plasmids[5]. This is a crucial quality since the whole processes of transformation requires
the bacteria to uptake plasmids and go through a genetic change.
2 Which is also called E. Coli 3 Warm-blooded animals 4 A type disease on the digestive systems 5 An outer layer, or barrier, of the bacteria that controls materials from entering
Seo Young Myaeng Candidate Number: 002213-064
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1.3 pUC18 Plasmid
Figure 3: Image of plasmid and bacterial DNA of a bacterium[5]
A plasmid is a circular double-stranded DNA molecule that is usually used in genetics.
Plasmid can be classified into two types: natural and artificial. The type of plasmid that will
be used in this investigation is pUC18, an artificial or genetically engineered plasmid.
Figure 4: Image of pUC18 plasmid molecule[6]
As shown in figure 4, pUC18 contains different types of genetic information, including one
for lacZ6. The lacZ and lac promoter
7 allow pUC18 to recombine and transform the genetic
information of a foreign DNA[7]. More importantly, pUC18 contains amp
R 8 and thus is
resistant to Ampicillin9. This character is essential to this investigation since the whole
transformation will be focused on making the bacteria resistant to Ampicillin. The existence
of ampR will allow the plasmid to be uninfluenced by the Ampicillin extant in LB-Ampicillin
plate[8].
6 A type of reporter gene, or gene that is easy to monitor 7 Promoter of lacZ 8 an antibiotic resistant gene 9 Antibiotic used to treat bacterial infection
Seo Young Myaeng Candidate Number: 002213-064
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1.4 History of Transformation
Transformation[8] is a process that can alter the genetic information of an organism. It can be
carried out by adding plasmid to the DNA that needs to be altered. Then, the two types of
DNA will recombine to form new genetic information.
Frederick Griffith’s experiment[9] was the prototype of the transformation process. In 1928, he
presented that harmless Streptococcus pneumoniae bacteria could be turned lethal through
exposure to heat-killed virulent strains. Through this, Griffith hypothesized that a factor from
the heat-killed virulent strains turned the Streptococcus pneumonia harmful.
Figure 5: Diagram of Frederick Griffith’s experiments [9]
After Griffith, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered that DNA
was responsible for making the bacteria strain transform[8]. They named the process of intake
of DNA by bacteria “transformation.”
Seo Young Myaeng Candidate Number: 002213-064
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2.0 Hypothesis
The purpose of this investigation is to determine the effect of the divalent cations’ size on
transformation efficiency. In order to change the cations’ size, cations of different atomic
mass will be used. This is because atomic mass is determined by the sum of the number of
protons and the number of neutrons10
that compose the atoms.
Atomic Mass
Mg2+
24.3
Ca2+
40.1
Mn2+
54.9
Table 1: the different divalent cations and their atomic mass
During transformation, a plasmid must enter the bacteria in order to change their genetic
information. However, because both plasmid and the bacteria cell membrane are negatively
charged, the plasmid will be repelled and prevented from entering. Thus, transformation will
less likely to be successful. However, when a chloride solution is mixed with bacteria, it will
dissociate into cations and chloride ions. The cations will neutralize the negative charges on
bacteria cell membrane. Therefore, the plasmid will no longer be repelled and can enter the
bacteria to change the genetic information, hence leading to successful transformation.
Figure 6: Diagram of how plasmid goes through plasma membrane when cations are used
In this investigation, because all three kinds of cations have the charge of 2+, they will be
able to neutralize the barriers to similar extents. This would mean in all transformation
processes, the plasmid will be able to successfully enter the bacteria. Thus, it can be deduced
that despite the different sizes of the three cations, the transformations will be successful.
10 Both protons and neutrons are (sub-)particles that make up an atom
Plasma
Membrane
Cations neutralize
the negative charges
Seo Young Myaeng Candidate Number: 002213-064
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3.0 Variables
3.1 Manipulated Variable
The atomic size of divalent cations, which will be altered by using cations of different
atomic mass (Ca2+
, Mg2+
, Mn2+
)
In order to use cations of different atomic sizes, 0.1M chlorides of cations with
different atomic masses – CaCl2, MgCl2, and MnCl2 – will be used.
3.2 Responding Variable
The transformation efficiency of E. Coli strain
After successful transformation, the bacteria will be able to survive on a LB-
Ampicillin plate, an antibiotic environment. By using the following method, transformation
efficiency for bacteria transformed with different cations will be obtained:
11
11 Refer to Appendix 1 for further information
Seo Young Myaeng Candidate Number: 002213-064
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3.3 Constant Variables
- Type of bacteria used:
In all transformation processes, E. Coli strain from the same bacteria stock will be used.
- Type of plasmid used:
For all trials of transformation, 5µl of pUC-18 plasmid will be used.
- Type of Ampicillin used:
The same antibiotic Ampicillin will be used to make the LB-Ampicillin plates.
- Concentration of chlorides:
The concentrations of all chlorides will be kept constant to 0.1M.
- Period of Incubation:
After transformation, all plates with the bacteria will be incubated for 48 hours.
- Temperature of Incubation:
The incubation temperature will be kept constant to 37°C, which is within the ideal range of
temperature for bacteria survival.
Seo Young Myaeng Candidate Number: 002213-064
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4.0 Materials and Methods
4.1 Overall Methodology
Plates (4.2)
Salt Solutions (4.3)
Culturing Bacteria Stock (4.4)
Preparation of Chlorides
Transformation (4.5)
Streaking
Leaving at room temperature (15min)
Inserting Luria broth (250µl)
Ice shock (1min)
Heat shock (42 , 90sec)
Ice shock (15min)
Inserting plasmid (5µl)
Ice shock (5min)
Inserting bacteria into chlorides (250µl)
Preparation of CaCl2, MgCl2, MnCl2 of the same concentration
Culturing E. Coli bacteria stock
Preparing salt solutions
Preparing LB and LB Ampicillin Plates
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4.2 Preparation of LB and LB-Ampicillin Plates
250ml of LB agar is prepared12
The table below shows processes to prepare either LB or LB-Ampicillin plates
LB Plates LB-Ampicillin Plates
Agar is evenly poured into petri dishes Agar is cooled until 57
4ml of Ampicillin is added to the agar
Agar is slowly swirled for Ampicillin to mix
Agar is poured evenly into petri dishes
After agar solidifies, dishes are closed and turned upside down
The dishes are stored in refrigerator at 4
4.3 Preparation of Salt Solutions
Serial dilution is used to prepare 0.1M of CaCl2, MgCl2, and MnCl213
4.4 Culturing Bacteria Stock
200µl of LB broth is prepared14
Using a sterile loop, a colony of E. coli is scooped and added to the LB broth
The loop is twirled to assure they are mixed well
The LB broth is incubated at 37°C for 24 hours
Using a new sterile loop, small amount of the LB broth is spread on a LB plate
The plate is closed and sealed using parafilm, and is incubated at 37°C for 24 hours
12 Refer to Appendix 2 for further information 13 Refer to Appendix 3 for further information 14 Refer to Appendix 4 for further information
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4.5 Transformation
Label 6 microcentrifuge tubes “+plasmid” and three tubes “–plasmid”
Using micropipette, 250µl of ice-cold CaCl2, MgCl2, and MnCl2, is each added to two
“+plasmid” tubes and one “-plasmid” tube
Using sterile loop, remove a colony of E. Coli from bacteria stock15
Add the colony to CaCl2 of a “+plasmid” tube
Using vortex, assure the solution is mixed well
Using centrifuge, suspend the solution down
Steps 3 to 6 are repeated for the other 8 tubes
All tubes are placed in ice for 5 minutes16
Using micropipette, in each “+plasmid” tube, 5µl of plasmid is added and mixed well
All tubes are left in ice for 15 more minutes
All tubes are removed from ice and are placed at 42 water bath for 90 seconds17
; it is
assured no water enters the tubes
Immediately, all tubes are returned to ice for 1 minute18
All tubes are removed from ice
Using micropipette, 250µl of Luria broth is added to every tube
The tubes are left at room temperature for 15 minutes
Using micropipette, 100µl of each tube’s solution is placed on a LB-Ampicillin plate
Using micropipette, 100µl of each “-plasmid” tube’s solution is placed on a LB plate
Using sterile loop, the solutions on the plates are streaked19
All plates are closed, turned upside down, and incubated at 37 for 48 hours
15 Bacteria stock from step “4.4 – Culturing Bacteria Stock” 16 Process of ice-shock 17 Process of heat-shock; used to open pores of bacteria membrane so that plasmid can enter the cell 18 Process of ice-shock; used to allow membrane pores to close and prevent plasmid from escaping 19 Refer to Appendix 5 for further information
Seo Young Myaeng Candidate Number: 002213-064
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5.0 Data Collection
5.1 Raw Data
5.1-1 Qualitative Data
Note: The following images are bacteria plates after 48th
hour of incubation
MgCl2
MgCl2 Plate 1 MgCl2 Plate 2
Plate Number Plate 1 Plate 2
Number of Colonies (CFU) 28 4
Observations It can be easily noticed that the plates have small
numbers of bacteria colonies. The colonies are also
small and cannot be seen unless observed closely and
carefully.
CaCl2
CaCl2 Plate 1 CaCl2 Plate 2
Plate Number Plate 1 Plate 2
Number of Colonies (CFU) 150 172
Observations The number of colonies formed on the two plates is
great. Most colonies are thick and big enough to be seen
easily.
Seo Young Myaeng Candidate Number: 002213-064
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MnCl2
MnCl2 Plate 1 MnCl2 Plate 2
Plate Number Plate 1 Plate 2
Number of Colonies (CFU) 80 152
Observations Many colonies have formed on the plates. Most
colonies are thick and big enough to be seen easily.
Negative Control
LB-Ampicillin Plates LB Plates
Type of Plate LB-Ampicillin LB
Type of
Chloride
MgCl2 CaCl2 MnCl2 MgCl2 CaCl2 MnCl2
Number of
Colonies
- - - N/A N/A N/A
Observations No colonies formed at all
on the plates
Though it is difficult to observe from the
image, a countless number of bacteria
colonies were formed. The plates are full of
streaks of bacteria colonies
Seo Young Myaeng Candidate Number: 002213-064
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5.1-2 Quantitative Data
Number of Bacteria Colony (CFU20
)
Plate 1 Plate 2 Average
MgCl2 28 4 16
CaCl2 150 172 161
MnCl2 80 152 116
Negative Control
(LBA plate)
- - -
Negative Control
(LB plate)
N/A N/A N/A
Table 2: Number of bacteria colonies on each plate on the 48th
hour
Note:
“-“: no colonies formed
“N/A”: the colonies were uncountable
20 Colony-forming unit: unit for measurement of bacterial number
Seo Young Myaeng Candidate Number: 002213-064
pg. 17/36
5.2 Calculation of Transformation Efficiency
The equation for transformation efficiency is:
For successful calculation, the total mass of plasmid used needs to be found out as below:
Where:
µl
The value of “total mass of plasmid used” is unknown, but can be found as shown below:
Thus:
µg
Therefore:
Chloride Average number of colonies
(CFU)
Transformation Efficiency (CFU/µg)
MgCl2 16
CaCl2 161
MnCl2 116
Table 3: Calculation of transformation efficiency for different chlorides
Seo Young Myaeng Candidate Number: 002213-064
pg. 18/36
5.3 Data Analysis
Figure 7: Diagram of transformation efficiency against type of divalent cations
0
50
100
150
200
250
300
350
Trn
asf
orm
ati
on
Eff
icie
ncy
/CF
U µ
g-1
Type of Cations
Transformation Efficiency/colonies µg-1 against Type of Cations
Magnesium
Calcium
Manganese
Magnesium Calcium Manganese
Seo Young Myaeng Candidate Number: 002213-064
pg. 19/36
Figure 8: Diagram of transformation efficiency against atomic mass
y = -0.7985x2 + 69.781x - 1192.1
R² = 1
0
50
100
150
200
250
300
350
0 10 20 30 40 50 60
Tra
nsf
orm
ati
on
Eff
icie
ncy
/CF
U µ
g-1
Atomic Mass of Cations
Transformation Efficiency/colonies µg-1 against Atomic Mass
Seo Young Myaeng Candidate Number: 002213-064
pg. 20/36
6.0 Statistical Analysis
6.1 ANOVA Test
ANOVA (Analysis of Variance) Test21
is one of the ways to compare the resulting data from
different cations and observe whether there is a significant difference in the results or not. For
the calculations, raw data on the number of colonies was used.
Null Hypothesis (H0): there is no significant difference among different chlorides
Alternate Hypothesis (HA): there is significant difference among different chlorides
If F ratio > F critical = Null Hypothesis is rejected and Alternate Hypothesis is made valid.
Note: Calculations were done using the Excel Program.
Source of Variation Sum of
Squares
df Mean squares
(s2)
F Ratio Critical F P Value
Between Groups 22,033
2 11,016
10.59 9.55 0.0440
Within Groups 3,122 3 1,040
Total 25,155
5
Table 4: Results of the ANOVA Test
Because F ratio (10.59) > F critical (9.55), the Null Hypothesis is rejected and the Alternate
Hypothesis is accepted.
Therefore, there is significant difference between groups.
21 Refer to Appendix 6 for further information and calculation
Seo Young Myaeng Candidate Number: 002213-064
pg. 21/36
6.2 Tukey’s HSD Test
The Tukey’s HSD (Honestly Significant Difference) Test22
can also be used to analyze
results of this investigation. This test will compare all possible pairs of groups to see which
group pair is greater than the critical value.
Tukey’s HSD Test Critical Value:
Where
Cations Atomic Mass Mean Difference
(of Average CFU)
Critical Value
135
135
135
Table 5: calculation and presentation of mean difference and critical value
In the ANOVA Test, results show that there is significant difference between groups.
However, this was when putting all three results together. The Tukey’s HSD Test directly
compares two cations at a time.
According to the test, the following pair has a mean difference greater than critical value.
Simultaneously, the fact that the pair of are close to the critical
value could also be considered (Then both pairs include the cations, magnesium.).
22 Refer to Appendix 7 for further information and calculation
Seo Young Myaeng Candidate Number: 002213-064
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7.0 Evaluation
7.1 Explanation
The results of this investigation were that transformation efficiency from transformation
using calcium cations was the greatest, manganese cations the second greatest, and
magnesium cations the lowest. Though the values of transformation efficiency are not equal
for the different sized cations, all transformations for different cations were successful. Thus,
it can be claimed that despite atomic size of cations, as long as they are divalent,
transformation will successfully occur. This can be explained by the fact that all three cations
have the same charge of 2+ and the same role of neutralizing bacteria membrane for plasmid
to enter the bacteria. Because of these common qualities, all cations are able to neutralize the
membrane to a similar extent and thus allow successful transformation to occur.
According to the ANOVA Test, the transformation efficiencies among different cations have
significant differences. However, the ANOVA Test compares the results from all three
groups simultaneously while the Tukey’s HSD Test compares two groups at a time. The
Tukey’s HSD Test states that there is no significant difference between calcium and
magnesium’s transformation efficiencies. Also, the mean difference between magnesium and
manganese is smaller than but close to the critical value. Thus, the possibility of experimental
errors behind the results of manganese or magnesium cations may be considered. These
statistics help show that the transformation efficiencies for divalent cations of different
atomic sizes are not of great difference and that atomic size may not be significant cause of
different transformation efficiencies.
Therefore, the results of this investigation can support the explanation and claim that
regardless of the sizes of the cations, as long as it is divalent, successful transformation will
occur.
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pg. 23/36
7.2 Uncertainties and Limitations
Amount of stock bacteria added: CaCl2, MgCl2, and MnCl2 were each mixed with a
colony of E. Coli scooped from the stock bacteria. Though it was attempted to keep the
amount of bacteria constant, the specific amount remains different. This difference in
amount of bacteria may cause significant difference in amount of transformed bacteria
and also in the amount of bacteria spread on LB-Ampicillin plate.
Amount of bacteria plated: It cannot be certain that the amount of bacteria plated after
transformation were equal. Since bacteria have the tendency to clump together, the 100µl
of plated solution may not have contained equal amount of bacteria. If so, then the
number of bacteria on the LB-Ampicillin plates would have differed and hence
influenced the overall result of the investigation.
Contamination of bacteria during transformation: During the transformation process,
the bacteria solution was exposed to open air and could possibly have been contaminated.
This means that other bacteria from open air may have been transformed together. When
counting the number of colonies transformed, the contaminant may also have been
counted together and affect the result of the investigation. Also, the bacteria solution may
have been contaminated because of long exposure to open air while being plated on LB-
Ampicillin plate. If the contaminant is resistant to Ampicillin, then it would have been
incubated and counted together with E. Coli bacteria, thus affecting the overall result.
Condition of the chlorides: The MgCl2 and MnCl2 used during the investigation were
man-made. Therefore, there is a possibility that an error occurred when creating the
chlorides. More error could have occurred if the magnesium or manganese did not
dissolve in the chlorides well and hence are not mixed well within the solutions. Then,
unequal amounts of cations would have been used during the investigation, and hence
influence the overall results.
Seo Young Myaeng Candidate Number: 002213-064
pg. 24/36
Condition of LB-Ampicillin Plates: The conditions of different plates used to spread
transformed bacteria could influence the results, too. The LB-Ampicillin plates used
were manmade and there is the possibility of the LB-broth not being mixed thoroughly.
If so, each plate would contain different amount of LB broth, or bacteria food. Then the
bacteria would have been incubated in “different environments” and influence the results.
Lack of trials and variables: In this investigation, there were only three manipulated
variables and for each variable only two LB-Ampicillin plates were created. The increase
in number of manipulated variables would have allowed better support and
understanding of results. It will also show whether the change in atomic size truly has a
specific pattern of relationship with transformation efficiency. Moreover, with greater
number of trials for each variable, there would have been more results to decrease the
experimental error and allow better comparison of the results.
Seo Young Myaeng Candidate Number: 002213-064
pg. 25/36
7.3 Ways to Improve
Making of chlorides: In this investigation, CaCl2 was a pre-made solution while the
other two were handmade. The condition of CaCl2 could not be controlled but is most
likely to have been stable. However, because both MgCl2 and MnCl2 were handmade
solutions, it is possible for the cations to not have been mixed well with the chloride.
Hence, the solutions would not have been as stable as CaCl2. The investigation may be
improved by assuring the chemicals are mixed well and are made into homogenous
solutions.
Increase in number of trials: In this investigation, because only two trials were done
for each cation, it is unsure whether the results are experimental errors or are actual valid
values. With greater number of trials, it will be possible to compare the results from the
different trials and evaluate whether the results are valid and reliable.
Increase in number of variables: In this investigation, there were only three
manipulated variables. Though a conclusion can be drawn with these results, if more
manipulated variables were used, then the results could turn out different. The increase in
number of variables would have allowed clearer views and understandings of the results,
and added to the validity and reliability of the investigation.
Adding similar amounts of bacteria: During the transformation process, several errors
occurred from lack of caution when adding bacteria. With more caution and care, these
errors can be improved and thus obtain more reliable results. For example, when adding
stock bacteria to the chlorides, with thorough measurement of the size of the colony
being added, error from the difference in amount of bacteria added can be decreased.
Also, by the end of the experiment, LB broth mixed with transformed bacteria has to be
added on LB-Ampicillin plate using micropipette. In this process, by simply mixing the
solution again before plating, it can be assured that the bacteria is evenly mixed within
the solution and thus be able to plate similar amount of bacteria on each plate.
Seo Young Myaeng Candidate Number: 002213-064
pg. 26/36
7.4 Further Investigation
Gene transformation is not a technique that can only be applied to bacteria. It is an efficient
method to change the genetic information of other organisms for the benefits of humans. For
example, currently, maize crops’ genetic information is being changed to increase crop yield.
Gene transformation can also be used on other organisms to gain information on genetic
diseases or ways to improve certain diseases. Because the technique can be used for various
reasons, it is crucial for efficient gene transformation to occur. In this investigation, three
different variables were used to determine whether different atomic size of divalent cations
affect transformation efficiency. The result was that despite the atomic sizes, as long as the
cations are divalent, transformation will occur. Through further investigations on this
experiment, other variables necessary for successful transformation may be found.
One further investigation that could be carried is determining whether the same rule applies
for bacteria other than E. Coli. This investigation was a success due to the usage of E. Coli,
but it does not mean that the hypothesis is true for all types of bacteria or organisms. It is
possible that different results will come out for different bacteria. It is possible to carry out
the same investigation as this one but alter the E. Coli into different organism. Thus the
different effects of transformation on different bacteria and organisms may be found out.
Another possible further investigation that could be carried out would also be to determine
the relationship between change in cations’ size and transformation efficiency. The only
difference would be that the cations will have a different charge. During this specific
investigation, all cations were divalent or had the charge of 2+. However, it is unknown
whether similar observations can be made for cations of different charges, such as 1+ and 3+.
Thus, it will be possible to determine whether the same observation made in this investigation
will be applicable for cations of different charges. Through this further investigation, the
effects of cations’ charge on transformation efficiency may also be determined. This would
be done by comparing the results from the further investigation and from this specific
investigation.
Seo Young Myaeng Candidate Number: 002213-064
pg. 27/36
8.0 Conclusion
During bacteria gene transformation, a chloride with divalent cations is added to make the
bacteria’s cell membrane more permeable to the plasmid. The plasmid, which enters the
bacteria cell will then be able to alter the genetic information of bacteria and hence make it
go through transformation. Normally, calcium chloride is used during transformation. But in
this investigation, chlorides of divalent cations with different atomic mass were used to
investigate the relationship between the atomic size and transformation efficiency.
Through the experiments, it was observed that though the values of transformation efficiency
were different for different divalent cations, transformation still occurred. The explanation
behind this is that because all three cations had equal charge of 2+, they were able to
neutralize the bacteria membrane to a similar extent to allow plasmid to enter the cell. Thus,
it can be claimed that despite the atomic size of cations, as long as it is divalent,
transformation will be successful.
Seo Young Myaeng Candidate Number: 002213-064
pg. 28/36
9.0 Appendix
Appendix 1) Calculation of “amount of plasmid plated in
”
Appendix 2) Preparation of LB agar
Using an electronic balance, 2.5g of tryptone, 1.25g of yeast, 2.5g of NaCl, and 3.75g
of agar are measured and prepared
The reagents are added to a 250ml conical flask along with 250ml of distilled water
The flask’s mouth is covered with a small beaker
The flask is placed inside an autoclave and is autoclaved until 17 psi
The autoclave is turned off and is left until the pressure is released
The conical flask is removed from the autoclave and is slowly swirled in order to
prevent the agar from solidifying
Appendix 3) Process of 10-fold serial dilution
Figure A) Diagram of 10-fold serial dilution
Appendix 4) Preparation of LB broth
10g of Luria-Bertani powder are added to 400ml of distilled water
The solution is mixed well and is autoclaved
10cm3 distilled water
100ml of 1M
Cacl2, MgCl2,
or MnCl2
0.1M
Seo Young Myaeng Candidate Number: 002213-064
pg. 29/36
Appendix 5) Method of Streaking
Solutions and/or bacteria are streaked on to plates as following[10]:
Figure B) Diagram on method of streaking bacteria
Seo Young Myaeng Candidate Number: 002213-064
pg. 30/36
Appendix 6) ANOVA Test [11]
The Analysis of Variance (ANOVA) Test allows a comparison of 3 or more groups, such as
the ones used in this experiment (Mg2+
, Ca2+
, and Mn2+
). By using the ANOVA Test, either
of the two following hypothesis will be accepted:
1. The Null Hypothesis,
This is when the change in cations’ atomic size, or atomic mass, does not cause change in
transformation efficiency of E. Coli.
2. The Alternate Hypothesis, one or more means are different
This is when it can be deduced that there is significant different between the groups’ results
and thus the change in atomic mass does bring change in transformation efficiency.
The ANOVA Test is also taken place with the following assumptions:
1. Results from one group are independent from the other; they do not affect the other
groups’ results.
2. Observations from each group follow the normal distribution statistics.
3. Variances of all groups are equal
Seo Young Myaeng Candidate Number: 002213-064
pg. 31/36
Below is an example of an ANOVA Test table and its summary:
Source of
Variation
Sum of
Squares (SS)
df Mean squares
(s2)
F
Ratio
Critical F P Value
Between
Groups
SSb
(computer
generated)
Within
Groups
SSw
Total SSt
Table A) Table on summary of ANOVA Test
Notation Meaning
SSb Sum of squares between groups
SSw Sum of squares within groups
SSt Sum of squares in total
df Degree of freedom
k Total number of groups (= 3)
N Total number of results (= 6)
Mean squares / Variance between groups
Mean squares / Variance within groups
Table B) Legend for Table A
Mg Ca Mn
x x2 x x
2 x x
2
Plate 1 28 784 150 22500 80 6400
Plate 2 4 16 172 29584 152 23104
32 322 232
800 52084 29504
Total Sum of
586
Total Sum of
82388
Table C) Calculation Step for ANOVA Test
Seo Young Myaeng Candidate Number: 002213-064
pg. 32/36
Calculation of :
Calculation of :
Calculation of :
Calculation of
Calculation of :
Calculation of F ratio:
F Critical Value of
Note: value was achieved from table below where and = .
Seo Young Myaeng Candidate Number: 002213-064
pg. 33/36
Table B) the F value or F distribution of ANOVA test [
12]
Seo Young Myaeng Candidate Number: 002213-064
pg. 34/36
Appendix 7) Tukey’s HSD Test[13]
Tukey’s HSD Test Critical Value:
Where
The HSD critical value will be compared with the differences of colony numbers between
two cations results. If the differences are larger than the critical value, then it can be claimed
that the results’ differences are significant.
Table C) Table of q values[14]
Note: df error represents the value
Seo Young Myaeng Candidate Number: 002213-064
pg. 35/36
Graphical Interpretation of Tukey’s HSD
Figure C) Graph of cations pair greater than critical value
0
50
100
150
200
Mg-Ca Mg-Mn Ca-Mn
Mean D
iffe
rence
Cations Pair
Mean Difference Above Critical Value
Mean Difference
Critical Value
Seo Young Myaeng Candidate Number: 002213-064
pg. 36/36
10.0 References
[1] http://biochemistry.yonsei.ac.kr/biochem_molecular/gene_cloning_17.php
[2] http://www.pnas.org/content/101/42/15027/F2.expansion.html
[3] “E. Coli Genotypes.” Wikipedia. 2011. Web.
<http://openwetware.org/wiki/E._coli_genotypes#DH5.CE.B1>.
[4] “Escherichi Coli.” Wikipedia. 2011. Web. <http://en.wikipedia.org/wiki/Escherichia_coli>.
[5] http://en.wikipedia.org/wiki/File:Plasmid_(english).svg
[6] http://www.mun.ca/biology/scarr/Plasmid_pUC18.html
[7] “Plasmid pUC18.” Memorial University. 2010. Web.
<http://www.mun.ca/biology/scarr/Plasmid_pUC18.html>.
[8] “Transformation (genetics).” Wikipedia. 2011. Web.
< http://en.wikipedia.org/wiki/Transformation_(genetics)>.
[9] https://filebox.vt.edu/users/mahogan2/Filebox%20Portfolio/Webquest%20for%20DNA_fil
es/image002.jpg
[10] http://www.marine.csiro.au/microalgae/methods/support%20or%20original%20files/micro
a3.jpg
[11] Yu-Xann, Jorrel Too. Determining the optimum temperature that produces the highest
transformation efficiency rate using the heat shock transformation method on
modified Escherichia coli strain. 2008. Print.
[12] http://www.velocebit.com/Courseware/Statistics/Common%20Files/Analysis%20of%20Va
riance/Text%20Figures/Table%20A6001.jpg
[13] Yu-Xann, Jorrel Too. Determining the optimum temperature that produces the highest
transformation efficiency rate using the heat shock transformation method on
modified Escherichia coli strain. 2008. Print.
[14] http://www.tcnj.edu/~ruscio/Guide%20to%20SPSS%20for%20Windows%20S11.pdf