IB Biology EE on Bacteria transformation by heat shock method using plasmid

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


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)


pg. 3/36

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


pg. 4/36

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+


pg. 5/36

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


pg. 6/36

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


pg. 7/36

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.”


pg. 8/36

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


pg. 9/36

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


pg. 10/36

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.


pg. 11/36

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


pg. 12/36

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


pg. 13/36

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


pg. 14/36

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



Observations The number of colonies formed on the two plates is

great. Most colonies are thick and big enough to be seen

easily.


pg. 15/36

MnCl2

MnCl2 Plate 1 MnCl2 Plate 2



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


pg. 16/36

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


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


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


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


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


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


pg. 22/36

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.


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.


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.


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.


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.


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.


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


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


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


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


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 = .


pg. 33/36

Table B) the F value or F distribution of ANOVA test [

12]


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


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


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

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