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INTERNATIONAL BACCALAUREATE DIPLOMA PROGRAM EXTENDED ESSAY BIOLOGY Determining the optimum temperature that produces the highest transformation efficiency rate using the heat shock transformation method on modified Escherichia coli DH5 strain. by CHAEHYUN LEE Candidate Number: 002213-048 Word Count: 3, 636

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IB Biology EE on bacteria transformation using heat shock method

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Page 1: IB Biology EE on Bacteria transformation by heat shock method using plasmid

INTERNATIONAL BACCALAUREATE DIPLOMA PROGRAM

EXTENDED ESSAY

BIOLOGY

Determining the optimum temperature that produces the highest

transformation efficiency rate using the heat shock

transformation method on modified Escherichia coli DH5 � strain.

by

CHAEHYUN LEE

Candidate Number: 002213-048

Word Count: 3, 636

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Acknowledgement

My extended essay couldn’t have finished without the support from:

Mr. Lawrence, for guiding us and supporting us throughout the process.

Jason Rhim and Michael Shin for sacrificing their time to helping us.

Seo young Myaeng for supporting and encouraging me as my extended essay partner.

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Abstract

Transformation is the transferring of extracellular DNA into an organism. Most bacteria aren’t able to

transform naturally because they are unable to absorb plasmids naturally. Thus, in order to allow

bacteria to uptake DNA, heat shock method is necessary to artificially induce competence using a

sudden increase in temperature for a brief period of time. This research therefore attempts to

determine which temperature will allow for optimum transformation efficiency.

Four different temperatures were tested: 9°C, 30°C, 42°C, and 51°C. The bacterium of choice was the

modified Escherichia coli DH5 ∂ and the plasmid was pGREEN. Plasmid pGREEN allows bacteria to glow

in yellow-green color and to become ampicillin-resistant. These transformed ampicillin-resistant

bacteria are able to survive on LB-ampicillin (LBA) plates because they have resistance against ampicillin,

which kills bacteria. However, untransformed bacteria are unable to survive on LBA plates. Therefore,

any bacteria that were present on an LBA plate were said to be transformed. The transformation

efficiency was calculated based on the number of colonies present after incubation overnight.

At 9℃ and 51℃, transformation efficiency for the two temperatures was zero. At 30℃, the

transformation efficiency was calculated to be 150. At 42℃, transformation efficiency was 500. 42℃ had

the highest transformation efficiency. Further statistical analysis using ANOVA test and Turkey’s HSD

test reveals that the temperature of 42°C is more significant (p< 0.05) and hence more efficient

compared to 30°C.

Based on the evidence obtained during this investigation, it can be concluded that the optimum

temperature for heat shock method is 42℃. This temperature is high enough to activate most heat

shock proteins and low enough to prevent proteins from denaturing.

(274 words)

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Table of Contents ABSTRACT ................................................................................................................................................................ 3 CONTENTS .............................................................................................................................................................. 4 1.0 INTRODUCTION ............................................................................................................................................. 5 1.1 Rationale for Study .................................................................................................................................................. 5 1.2 Transformation ........................................................................................................................................................ 6 1.2.1 E. Coli DH5 � .............................................................................................................................................. 6 1.2.2 Plasmid PGREEN ........................................................................................................................................ 7 1.2.3 History of Transformation ...................................................................................................................................................... 8 1.2.4 Uses of Transformation ............................................................................................................................................................ 9 2.0 HYPOTHESIS .................................................................................................................................................. 10 3.0 VARIABLES ...................................................................................................................................................... 11 3.1 Manipulated Variable ............................................................................................................................................ 11 3.2 Responding Variable ............................................................................................................................................ 12 3.3 Controlled Variable ............................................................................................................................................... 13 4.0 METHODOLOGY ............................................................................................................................................ 14 4.1 General Overview of Methodology .................................................................................................................... 14 4.2 Preparation of LB: Amp plates ........................................................................................................................... 15 4.3 Preparation of Competent Bacteria ................................................................................................................... 15 4.4 Transformation ...................................................................................................................................................... 15 5.0 DATA COLLECTION ...................................................................................................................................... 18 5.1 Raw Data Collection ............................................................................................................................................. 18 5.1.1 Qualitative Data ........................................................................................................................................ 18 5.1.2 Quantitative Data ..................................................................................................................................... 19 5.2 Data Processing ................................................................................................................................................... 20 5.2.1 Calculation of Transformation Efficiency ............................................................................................. 20 6.0 STATISTICAL ANALYSIS ......................... ..................................................................................................... 22 6.1 ANOVA Test ........................................................................................................................................................... 22 6.2 Turkey’s HSD Test ................................................................................................................................................. 23 7.0 EVALUATION .................................................................................................................................................. 24 7.1 Explanation of Results ......................................................................................................................................... 24 7.2 Uncertainties and Limitations ............................................................................................................................ 25 7.3 Ways to Improve .................................................................................................................................................. 26 7.4 Further Investigations .......................................................................................................................................... 26 8.0 CONCLUSION ................................................................................................................................................ 28 9.0 APPENDIX ....................................................................................................................................................... 29 10.0 BIBLIOGRAPHY ........................................................................................................................................... 36

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1.0 Introduction

1.1 Rationale for Study

The transfer of new DNA into organisms has led to many improvements in the fields of science,

especially medicine. Thus, I found transformation to be a worthy topic of research. The technique of

transformation in plants and animals are extremely complex and costly. However, gene transfer in E.

Coli bacteria are relatively simple and suitable for school laboratories. Hence, E. Coli was chosen to go

through heat shock transformation procedure.

Although heat shock method is less costly than electroporation method1, which is another method to

induce competency 2of bacteria cells, heat shock

3 protocol is not inexpensive as well. Thus, it is crucial to

increase the efficiency of transformation as much as possible.

The heat shock method has several variations. I decided to investigate the impact of temperature on

transformation rate because the temperature directly affects the efficiency of heat shock, which is a

crucial part of transformation protocol. The temperatures of 37℃ and 42℃ are known to be the most

common and temperatures found in scientific sources. It is important to find out which temperature

works the best for transformation.

Thus, by determining the optimum temperature for the heat shock methodology, I can help contribute

to bacterial transformation research and to fellow students who have limited number of trials and

limited time.

Therefore, my precise research topic is:

Determining the optimum temperature that produces the highest

transformation efficiency rate using the heat shock transformation method on

modified Escherichia coli strain.

1 A method that uses electrical shock to create temporary competency within the cell.

2 The state of being able to uptake new genetic information

3 A method that uses heat shock to temporarily allow cell membrane to be permeable

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1.2 Transformation

1.2.1 E. Coli DH5 �

E. Coli DH5 ∂ is a harmless, rod-shaped bacterium [1]. One significant characteristic of E. Coli DH5 ∂ is

that it is a gram negative bacterial strain. Its gram negative structure is important in the process of

transformation because it allows transformation process to work.

As opposed to gram-positive bacteria, gram-negative bacteria have a thinner layer of peptidoglycan and

a periplasmic space between the cell wall and the membrane [2]. On top of that, gram-negative bacteria

have no nuclear membrane.

These characteristics allow gram-negative bacteria, E. Coli DH5 ∂ , to be highly transformable. Moreover,

since E. Coli DH5 ∂ is a relatively harmless bacterium, it can be handled safely during experiments.

Figure 1: Difference between the structure of gram positive and gram negative bacteria

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1.2.2 Plasmid PGREEN

Plasmid is a vector and a self-replicating circular strand of DNA that can be modified. Once the vector is

absorbed by the organism, certain proteins that are not normally synthesized will be present. These

proteins may confer antibiotic resistance.

The selectable marker of plasmid pGREEN is beta-lactamase. pGREEN’s color marker is mutant Green

Fluorescent Protein4 fusion gene. This allows transformed bacteria to glow in yellow-green color. The

phenotypes of pGREEN’s transformants are ampicillin-resistant and yellow-green colonies.

4 Green Fluorescent Protein (GFP) is a protein that exhibits bright greenfluorescence when exposed

to ultraviolet blue light.

Figure 2: Plasmid map of pGREEN

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1.2.3 History of Transformation

The idea of transformation was first introduced when

virulent5 strain of Streptococcus Pneumoniae

the same species [3].

Griffiths’ experiment paved a way for Avery, McCarty, and MacLeod to find out in 1944 that special

loops containing genetic information can be transferred from one bacterium to another. When cell

membrane was ruptured due to heat shock, DNA was released from heat

nonpathonogenic strain, which showed newly acquired gene. This phenomenon was known to be

transformation.

5 Harmless

6 Harmful

7 Virulent strain of bacteria

Figure 2: Griffith’s experiment and its results

Chaehyun Lee

History of Transformation

idea of transformation was first introduced when Frederick Griffiths discovered in 1928 that a non

Streptococcus Pneumoniae6 could turn virulent

7 when exposed to

experiment paved a way for Avery, McCarty, and MacLeod to find out in 1944 that special

loops containing genetic information can be transferred from one bacterium to another. When cell

membrane was ruptured due to heat shock, DNA was released from heat-killed Pneumoniae to

nonpathonogenic strain, which showed newly acquired gene. This phenomenon was known to be

s experiment and its results

002213-048

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Frederick Griffiths discovered in 1928 that a non-

when exposed to virulent strains of

experiment paved a way for Avery, McCarty, and MacLeod to find out in 1944 that special

loops containing genetic information can be transferred from one bacterium to another. When cell

lled Pneumoniae to

nonpathonogenic strain, which showed newly acquired gene. This phenomenon was known to be

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1.2.4 Uses of Transformation

Transformation has a wide range of uses. One of its earliest uses is harvesting human insulin from

transformed bacteria [4]. This is particularly useful because patients suffering from diabetes can have a

ready supply of insulin at hand. Since human insulin is used instead of previous sources such as sheep

and cattle, allergies will rarely occur.

Another notable use of transformation was made possible by the introduction of GFP, which is a protein

that allows bioluminescence [5]. The potential of transformation was acknowledged in the Chemistry

Nobel Prize Award in 2008. This GFP, found in jellyfish, creates bioluminescence, and glows almost

immediately when synthesized because it does not require a substrate. GFP has potential to allow non-

invasive diagnosis and protein tracing. For instance, if researchers want to know whether gene X is

involved in the formation of blood vessels, they can link gene X to the GFP protein. If the blood vesses

glow with GFP, it would be an indication that gene X is involved in blood vessel formation. Moreover,

when GFP is used in eukaryotic human liver cells, researchers can detect transfected cells by using the

glowing GFP [6].

Figure 3: Glowing jellyfish with GFP

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2.0 Hypothesis

Today, the widely accepted view is that most bacteria are incompetent because they are unable to

absorb plasmids directly from their surroundings. Hence, in order to artificially induce competency of

bacteria, heat shock method has to be used. The method is theorized to work as follows:

First, incompetent bacteria are subjected to heat shock, which is a sudden increase in temperature.

Then, HSPs8 are activated which cause pores on the membrane of bacteria to dilate or new pores to be

created. Then, as Calcium Chloride is introduced, Calcium ions (Ca2+

) 9 neutralize the negative charges

found on the cell surface of bacteria and DNA. This thereby allows a DNA molecule to adhere to the

surface of bacteria. Then, Chloride (Cl-) ions enter bacteria. This sudden influx of chloride ions creates a

thermal imbalance on either side of the cell membrane, which forces the plasmid DNA to enter the cells

through cell pores. Bacteria become competent.

The most crucial step above is to make sure that Heat Shock proteins are activated because Heat Shock

Proteins activate reactions that eventually lead to the artificial competency of bacteria.

It is very essential to find out the optimum temperature because this will allow the greatest percentage

of Heat Shock Proteins to be activated. If the temperature is too low, the heat shock proteins will not be

activated. However, if the temperature is too high, bacteria may die due to protein denaturization.

Note: The exact mechanics of transformation is unknown. Thus, the above hypothesis is purely

theoretical although widely accepted.

8 Heat shock proteins, which are expressed when cells are suddenly exposed to sudden increase in temperature

9 Cation that allows the entrance of plasmids through cell membrane

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3.0 Variables

3.1 Manipulated Variable: Temperature used for heat shock method

Heat shock treatment is vital in transformation of bacteria because most bacteria are unable to directly

absorb the plasmid from their surroundings. Thus, bacteria have to experience sudden increase in heat

so that their plasma membrane would open and allow plasmids to be absorbed.

To measure the temperatures accurately, digital Logger Pro temperature probe device was used.

The following is a desired list of different temperatures that will be used for heat shock.

Temperature/ ℃ Expected qualitative observation

9.0 No transformation should occur because

bacteria wasn’t subject to sudden increase in

heat. Heat shock proteins are not expected to be

activated.

30.0 Some colonies of bacteria will be transformed

and appear on LBA 10

plate due to change in

temperature. However, the number of colonies

is not expected to be as many as that of a higher

temperature (42. 0℃).

42.0 Majority of bacteria will be transformed because

they will effectively absorb plasmid due to

change in heat.

51.0 No transformation should occur because most

proteins will be denatured.

Table 1: Range of temperature for heat shock

10

Luria Broth- Ampicillin. Non-transformed bacteria cannot survive in LBA conditions.

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3.2 Responding Variable: The CFUs11

formed on the LBA plate at the end of experiment,

represented as the transformation efficiency rate.

Ampicillin contains antibiotic properties. Thus, when bacteria that have gone through transformation

are plated on a LB-ampicillin plate, only the ones that are resistant to ampicillin can survive and form

colonies. However, bacteria that are not transformed have no resistance to ampicillin and thereby can’t

survive on LBA plate.

The surviving bacteria colonies will be counted and recorded. Transformation efficiency rate will be

measured using the following calculations.

Step 1: Find the total mass of plasmid in fraction

Mass of plasmid used x ���� � � �������� � ��� � ����

� �� � ���� � �������� �

Step 2: Calculate transformation efficiency

Transformation efficiency= ������ � � ����

� �� ��� � ������ �� ���� �

11

Colony Forming Units (CFU) is a measure of viable bacterial numbers.

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3.3 Controlled Variables

� Type of bacteria: Escherichia Coli DH5 �

Modified E. Coli will be used throughout the experiment. This modified stain of E. Coli is relatively safe

and is not pathogenic. Contamination will be prevented by tightly sealing the plate and the bottle that

contains E-coli. Contamination can also be reduced by not talking (opening mouth to let out bacteria)

while performing transformation.

� Type of Plasmid: PGREEN12

Plasmid P-Green will be used throughout the entire experiment.

� Antibiotic: Ampicillin

Ampicillin will be used to test the transformation efficiency of bacteria. Ampicillin will be poured in LB to

create LB-ampicillin plate. Ampicillin also prevents contamination from external source because bacteria

that are not competent to ampicillin cannot survive.

� Incubation method

To ensure that bacterial growth is systematic throughout the entire experiment, incubation period and

temperature will be kept constant. Period is for 24 hours and temperature is 37 ℃.

12

See Appendix 2

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4.0 Methodology 4.1 General Overview of Methodology

Chaehyun Lee

ethodology

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9℃

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4.2 Preparation of Ampicillin-LB agar (LBA) plate

1. Melt 500ml of LB agar13

in a microwave.

2. 0.300mg of Ampicillin is added to LB agar when the temperature of LB is dropped down to

57.0 ℃.

3. Mixture is mixed thoroughly and poured into petri dishes.

4. The dishes are left at room temperature (37℃) for 2 minutes until LB is solidified.

5. LB plates are turned upside-down. *This is to prevent water vapor from dropping on LB agar.

6. LB plates are stored in a refrigerator.

4.3 Preparation of Competent Bacteria

1. Four 1.5ml microcentrifuge tubes are prepared and are labeled: 30℃, 42℃, 51℃, 9℃.

2. 250ul of ice-cold Calcium Chloride is placed in each microcentrifuge tube.

3. Using a sterilized loop, a single colony of E.coli culture is placed in each microcentrifuge.

4. Each mircrocentrifuge tube is stirred using vortex.

5. All microcentrifuge tubes are placed on ice for 5 minutes.

6. 5ul of plasmid pGREEN is added to each microcentrifuge by using a micropipette.

7. Microcentrifuge tubes are returned to ice for at least 15 minutes. Bacteria are ready to

undergo heat shock.

4.4 Transformation (Heat shock procedure)

4.4.1 At temperatures of 30℃, 42℃, 51℃

1. Waterbaths at temperatures 30℃, 42℃, 51℃ are prepared.

2. Each microcentrifuge is placed in a float and dropped into corresponding waterbath

(Microcentrifuge labeled 30℃ is placed in 30℃ waterbath). A stopwatch is started

immediately.

3. After 90 seconds, all microcentrifuges and floats from each water bath are removed at the

same time. They are returned to ice for 1 minute.

13

See Appendix 1 for preparation of method

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4. 250ul of LB is added to each microcentrifuge and are left at room temperature for 15

minutes.

5. 100ul of transformed cells are removed from one microcentrifuge by using micropipette and

are spread evenly on the surface of LB Ampicillin plate. This step is done for all three

microcentrifuges (30℃, 42℃, 51℃).

6. Petri dishes are turned upside-down and incubated at 37℃.

4.4.2 At temperature 4℃

1. The temperature of a refrigerator is set at 4℃.

2. Microcentrifuge labeled 4℃ is placed in the refrigerator. A stopwatch is started

immediately.

3. After 90 seconds, the microcentrifuge is removed from the refrigerator and is placed on ice

for 1 minute.

4. 250ul of LB is added to microcentrifuge and is left at room temperature for 15 minutes.

5. 100ul of transformed cells are removed from the microcentrifuge by using micropipette and

are spread evenly on the surface of LB Ampicillin plate.

6. Petri dish is turned upside-down and incubated at 37℃.

Diagram 1: Example of heat shock transformation method

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4.4.3 Collection of Results

1. After incubation for one night, the petri dishes are removed from the incubator.

2. Petri dishes are placed on top of a black paper.

Figure 4: Inverted petri dish showing e-coli colony

3. Number of bacterial colonies is counted and noted.

4. This experiment is duplicated to reduce experimental error.

4.4-4 Observation of glowing pGREEN bacteria

1. After incubation, petri dishes that contain transformed bacteria are brought into a dark

room. There should be no presence of light.

2. Ultra violet light is shined directly above the petri dishes.

3. Glowing bacterial colonies are observed.

CFU

CFU

Figure 5: Glowing E-coli colonies under UV light

UV light

CFU

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5.0 Data Collection

5.1 Raw Experimental Data 5.1.1 Qualitative Data

Figure 9: Trial 1 CFU count of 42℃ (left) and Trial 2 CFU count of 42℃ (right)

[Left] It is possible to observe the production of 6 colonies.

[Right] It is possible to observe the production of 4 colonies.

Figure 6: Positive control (left) and negative control (right)

[Left] E-coli colonies thrived in LB plate.

[Right] There is no presence of any colony in LBA plate.

Figure 8: Trial 1 CFU count of 30℃ (left) and Trial 2 CFU count of 30℃ (right)

[Left] It is possible to observe the production of 3 colonies.

[Right] There are no observable colonies present on the plate.

Figure 10: Trial 1 CFU count of 51℃ (left) and Trial 2 CFU count of 51℃ (right)

[Left] There are no observable colonies present on the plate.

[Right] There are no observable colonies present on the plate.

Figure 7: Trial 1 CFU count of 9℃ (left) and Trial 2 CFU count of 9℃ (right)

[Left] There are no observable colonies present on the plate.

[Right] There are no observable colonies present on the plate.

CFU

CFU

Control

At 9℃

At 42℃

At 51 ℃

At 30 ℃

℃ At 9℃

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5.1.2 Quantitative Data

Temperature,

Number of

CFUs in Trial

1

Number of

CFUs in Trial

2

Average

number of

CFUs/ CFU

9 - (a) - -

30 3 - �� = 1.5

42 6 4 ��� = 5

51 - - -

Table 2: Average CFU at different temperatures

(a)= No CFU is found

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5.2 Data Processing 5.2.1 Calculation of Transformation Efficiency

Before calculating the transformation efficiency, the total mass of plasmid must first be found. The total

mass of plasmid can be calculated by using the formula below:

Total mass of plasmid= (Total mass of plasmid) X � !"#$%& %' ()(*+&($%& *)# %& *,!#+

-%#!, .%,)/+ %' ()(*+&($%&

Mass of plasmid used

= Concentration of plasmid X Volume of plasmid solution used

= 20ng/µl X 5µl

= 100ng

= 0.1µg

Fraction of suspension put on plate: 100µl

Total volume of suspension: 500µl

Total mass of plasmid in fraction = (0.1) X ���0�� = 0.02µg Transformation efficiency can now be calculated using the formula below:

Transformation efficiency= 1)/2+ %' "%,%&$+(

-%#!, /!(( %' *,!(/$3 $& ' !"#$%& Temperature, ℃ Number of

CFUs in Trial 1

Number of

CFUs in Trial 2

Average

number of

CFUs/ CFU

Transformation

efficiency

(colonies/ µg )

9 - (a) - - ��.�� =0

30 3 - �� = 1.5

��.��= 150

42 6 4 ��� = 5

���.��= 500

51 - - - ��.��= 0

Table 3: Transformation efficiencies for Trials 1 and 2 at different temperatures

(a) = No CFUs found

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Graph 1: Shows transformation efficiency against temperature

0

100

200

300

400

500

600

9 30 42 51

Tra

nsf

orm

ati

on

Eff

icie

ncy

/ co

lon

ies

μg

-1

Temperature/ oC

Transformation Efficiency/ colonies μg-1 against Temperature/ oC

Transformation Efficiency

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6.0 Statistical Analysis

6.1 ANOVA Test

In order to find out which of the temperatures done in this experiment is significant, an ANOVA test [7]

(Analysis of Variance Test) is done. Raw data collected is used for this statistical test.

The ANOVA test will determine whether null or alternate hypothesis will be accepted.

Null hypothesis (H0): No significant difference among different temperatures

Alternate hypothesis (HA): There is a significant difference among different temperatures.

Null hypothesis is accepted if F ratio < F critical.

Alternate hypothesis is accepted if F ratio > F critical.

Source of

Variation

Sum of

squares

Degree of

freedom

(df)

Mean

squares (s2)

F Ratio Critical F P Value

Between

Groups

33.3 3 11.1 6.81 6.59 (computer

generated)

Within

Groups

6.5 4 1.63

Total 39.8 7

Table 4: Results of the ANOVA Test

6.81(F Ratio) > 6.59 (F Critical) = Null hypothesis rejected and alternate hypothesis accepted.

There is a significant difference among different temperatures.

Heat shock treatment does have impact on transformation efficiency.

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6.2 Turkey’s HSD Test

A significant F ratio only shows that the aggregate difference among the means of the several samples is

significantly greater than zero. It does not show whether any particular sample mean significantly differs

from any particular other. Thus, Turkey’s HSD test will be done by comparing all possible pairs of groups

to determine which pair is greater than the critical value.

The critical value of Turkey’s HSD is found to be:

Critical Value = 3.62 4567�

= 3.62 4�.8��

= 3.27

Group combination

Temperature / ℃

Mean difference Critical value Implication

9 30 1.5 – 0 = 1.5 3.27 No Significant difference

9 42 5 – 0 = 5 3.27 Significant difference

9 51 0 – 0 = 0 3.27 No Significant difference

30 42 5 – 1.5 = 3.5 3.27 Significant difference

30 51 1.5 – 0 = 1.5 3.27 No Significant difference

42 51 5 – 0 = 5 3.27 Significant difference

Table 5: Comparison of mean difference and critical value

There is a significant difference in the mean number of CFUs between two different temperatures for

the below three sets. The following pairs have a mean difference greater than critical value.

There is a significant difference in the mean number of CFUs between:

o 9℃ , 42℃

o 30℃ , 42℃

o 42℃ , 51℃

Notice that all pairs involve the temperature of 42℃.

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7.0 Evaluation

7.1 Explanation of Results

From the results, transformation efficiency at 9℃ and 51℃ is zero.

Possible reasons for the result can be as following:

a. Cells were unable to take in plasmid and were therefore unable to synthesize beta lactamase

which hydrolyzes ampicillin.

b. Proteins in cells were denatured due to high temperature.

At 9℃, there was no sudden change in temperature which means that bacteria failed to go through

heat shock. Thus, HSPs were not activated. Pores on cell membrane were not stimulated and made it

impossible for bacteria to take in plasmids. Therefore, bacteria at 9℃ were incompetent and failed to

survive in antibacterial (LB- ampicillin) environment.

Another possible explanation is that the prolonged exposure to cold had damaged the bacteria and

thereby slowed down their growth. E.Coli hibernates when exposed to freezing temperatures. Hence,

when they were exposed to 9℃ conditions for an extended period, they might have started hibernation.

Hibernation prevents molecules from moving in and out of the bacteria. This results in no plasmid

uptake and therefore no antibacterial resistance.

At 51°C, proteins in cells were denatured due to high heat. Since important enzymes were unable to

maintain bacteria’s active site, reactions could no longer be catalyzed and bacteria were killed. HSPs,

which could have reformed the denatured protein, were also denatured. Plasmids are also very

vulnerable to high temperature. Thus, when they were subjected to 51°C, they denatured rapidly. The

high temperature had denatured both plasmids and bacterial proteins, which made it highly impossible

for transformation to occur, resulting in zero transformation efficiency.

At 30°C, transformed bacteria colonies were only present on one plate (trial 1). Results from

Turkey’s HSD test shows that this temperature doesn’t reach the critical value. Possible explanation for

the low efficiency is that the temperature increase was not high enough to activate HSP. It is known that

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HSPs are activated when cells undergoes stress, such as exposure to high temperature. However,

temperature at 30°C was not high enough to give stress to cells. Thus, HSPs remained inactivated and

allowed only small amount of plasmids to be taken in.

At 42°C, there was the highest transformation efficiency. 42°C was high enough to provide sudden

increase in temperature and activate HSPs. Thus, the most number of pores were created which

resulted in greater influx of plasmids and Cl- ions. Plasmids gave antibacterial resistance to E-coli colony

and allowed the most number of them to survive in LB-ampicillin conditions.

Therefore, heat shock is most effective at 42°C because this temperature is high enough to create pores

and keep it open for plasmids to enter but short enough to prevent denature of plasmids and bacterial

proteins.

7.2 Uncertainties and Limitations

The amount of bacteria used for each trial varied because it was impossible to take out the exactly same

amount of bacteria each time by using the loop. Although careful attention was paid to take out the

same size of colony each time, it is uncertain how much bacteria were transferred. This could have had

significant impact on the results because the greater amount of bacteria would result in higher chances

for transformation and vice versa.

Counting the number of colonies couldn’t be accurate because there is a chance that two colonies that

are placed next to each other are merged and are mistaken as one. This would lower the transformation

efficiency count because less number of colonies would be counted. Also, due to the limitations of the

sight of naked eyes, smaller sized colonies could have been overlooked or mistaken as air bubbles.

There is a possibility of contamination that could have possibly introduced new strands of bacteria that

are resilient to the antibiotics. Although it is assumed that ampicillin in the agar would kill any

contaminants, some bacteria may be resilient.

The final temperature cannot be conclusively said to be the optimum temperature because of lack of

trials and replicates. Because of the lack of plasmid pGREEN, this experiment was limited to duplication

rather that the preferred triplication. Since transformation has a high margin of error, two trials cannot

be enough to conclusively say that the results are accurate and precise.

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The range of temperatures used was very limited. In this experiment, only four different temperatures

were used due to lack of laboratory facilities. These four temperatures are not enough to draw a precise

conclusion because there is a wide range between two temperatures, such as 30°C and 42°C.

7.3 Ways to Improve

In order to control the amount of bacteria that are used in each trial, a more effective equipment should

be used such as rotating incubator. This equipment can help make sure that equal amounts of bacteria

go into each respective microcentrifuge tube by preventing the clumping of bacteria.

In the final steps of the experiment, electronic bacterial colony counter should be used to detect the

number of colonies with higher accuracy. Counting the number of colonies using naked eye (manual

detection) is less effective because smaller colonies, that are hard to see with naked eyes, may be

overlooked. Thus, an electronic bacterial colony counter will enhance the accuracy of the experiment by

reducing the human error.

A wider range of temperatures should be experimented to accurately determine the optimum

temperature. Since there is a big range between two temperatures, for example 30°C and 42°C, more

temperatures should be used to draw a more precise conclusion.

To further enhance the precision and accuracy of the experiment, more plates should be tested per trial.

During this experiment, only two plates were used. This is not suitable for transformation because

transformation technique has a high margin of error. Thus, more plates should be done and more trials

should be carried out (triplicate) and the average should be computed to get a more accurate data.

7.4 Further Investigation

Green Fluorescent Protein (GFP) used during transformation should be further applied to research as a

reporter molecule. A reporter molecule is one protein, such as GFP, linked to the protein that is subject

to study. By locating the protein with the reporter molecule, it becomes possible to follow what the

protein is doing. For an instance, if one wanted to know whether gene X was involved in the formation

of blood vessels, one can link gene X to the GFP gene. Then, the cells would make a protein that was X

plus GFP, resulting in fusion protein. Thus, if the blood vessel began to glow with GFP, it would be a hint

that protein X was involved in blood vessel formation.

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GFP has a great potential for the future of microbiology and other medical fields because it can also be

applied to eukaryotic cells such as human liver cells. Transfection will allow researchers to easily detect

transfected cells by using the glow of GFP. This method becomes highly useful when studying cancer and

neurobiology.

To increase transformation efficiency, other factors that might impact transformation should also be

studied. An example would be trying out different concentrations of Calcium chloride. Ca2+

ions

produced from CaCl2 are crucial factors in heat shock process. It is important to locate the effects of

increasing or decreasing the concentration of Calcium chlorides because it will allow the experiments to

not waste any amount of plasmid and improve the transformation protocol.

The impact of duration of heat shock on transformation efficiency should also be investigated. A longer

heat shock but at a lower temperature might produce more number of colonies than a brief heat shock

at a higher temperature. This is because the longer heat shock duration increases the chance of

exogenous plasmid entering the bacteria. This might lead to an alternate, even more effective,

transformation treatment.

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8.0 Conclusion

Based on the evidence obtained during this investigation, it can be concluded that the optimum

temperature for heat shock method is 42℃. This temperature is high enough to activate most heat

shock proteins and low enough to prevent proteins from denaturing. At 42℃, activated HSPs enlarged

and created pores on the bacterial cell membrane and significantly increased the uptake of plasmids.

Hence, the highest transformation efficiency was achieved.

At 30℃, transformation efficiency was not as high as that of 42℃ because 30℃ was not high enough to

induce most HSPs to be activated. Nonetheless, 30℃ condition was more efficient for heat shock than

other extremely low and extremely high temperatures such as 9℃ and 51℃. At lower temperature (9℃) heat shock proteins were not fully activated because there was no sudden

increase in temperature. In contrast, at higher temperature (51℃), bacteria died due to plasmid

denaturalization. In this condition, transformation may occur because HSPs are still activated but the

transformation efficiency cannot be high because the temperature is too high for bacteria too survive.

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9.0 Appendix

Appendix 1- Preparation of Luria Broth agar

Reagents:

2. 5 g – Sodium Chloride

3. 5 g – Tryptone

4. 7.5 g – Bacteriological Agar

5. 500 ml – Water

Methodology:

The reagents above are each measured using an electronic weighing machine and are all placed in a

beaker. 500ml of distilled water is added to the beaker. Heat the beaker using a Bunsen burner

while stirring the contents with a glass rod. When all reagents are homogenized, place them in a

pressure bottle and sterilize using autoclave machine. When still in hot liquid state, pour the LB agar

into petri dishes. Each petri dish should be about half-filled. Agar is left to cool.

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Appendix 2- ANOVA test

Null hypothesis H0 = u1= u2= u3= u4 means that the change in temperature doesn’t cause E-coli to have

greater transformation efficiency.

Alternate hypothesis is defined as HA= one or more means are different. This means that there is a

significant difference between groups. Thus, it can be concluded that the heat shock treatment does

have impact on transformation efficiency.

All significances for the following test will be at 5% of 0.05.

Assumption for the ANOVA test:

1. Observations are independent of each other. Colony formation for one result does not influence

the colony formation for another result.

2. The observations in each group conform to normal distribution.

3. Variances of all groups are homogeneous (equal).

The ANOVA test can be summarized as the table below:

Source of

Variation

Sum of

squares

Degree of

freedom

(df)

Mean squares

(s2)

F Ratio Critical F P Value

Between

Groups

SSb (k-1) MS�� =

66=�>?

MS� MS=

Fk-1, N-k ( computer

generated)

Within

Groups

SSw (N-k)

MS=� = 66=�>?

Total SSt (N-1)

Table 6: Summary of ANOVA calculations

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Conventional notation Meaning

SSb Sum of Squares between groups

SSw Sum of Squares within group

SSt Total sum of Squares

df Degree of Freedom

k Total number of groups

N Total number of results

@ABC Mean squares (variance) between groups

@ADC Mean squares (variance) within group

Table 7: Legend for ANOVA test

Temperature/ ℃ 9 30 42 51

X X2 X X

2 X X

2 X X

2

Result 1 - - 3 9 6 36 - -

Result 2 - - - - 4 16 - -

Σx

ΣΣx2

0 0 3 9 10 52 0 0

Table 8: Calculation for ANOVA

(-)= No CFUs found

Total summation Σx = 13

Summation for squared ΣΣx2 = 61

SSb = E6����� � � F℃)G������ � ����� H…… + E6����� � � 0�℃)G

������ � ����� H – (JK )G

� �� ������ � �����

= E�G� + �G

� + ��G� + �G

� H - ��G

M

= [ 0 + 4.5 + 50 + 0 ] – 21.1

= 54.5 – 21.2

= 33.3

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SSw can be found by ΣΣx2

– E6����� � � F℃)G������ � ����� … … + 6����� � � 0�℃)G

������ � ����� H

= 61 – 54.5

= 6.5

The total sum of squares (SSt) is SSw + SSb = 6.5 + 33.3

= 39.8

Calculating the mean squares/ variance

MS� = 66O��O

MS= = 667��7

= ��.�

� = 8.0P

= 11.1 = 1.63

The F ratio for this experiment is = 56O567

=

��.��.8�

= 6.81

F critical of F 3,4 = 6.59 (refer to the table below)

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Figure 11: Percentiles of F Distribution

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Appendix 3- Turkey’s Honestly Significant Difference (HSD)

HSD = q (α, k, N-k) 4QRS&

The value of α= 0.05 (represents the significant level)

k = 4 (represents the total number of groups)

N-k = 4 (Total number of results – total number of groups)

q = the value based on α, k, N-k

= 3.62

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Appendix 4- Graphical interpretation of Turkey’s HSD

Graph 2: Shows temperature pairs that are greater than the critical value

0

1

2

3

4

5

6

9 – 30 9 -42 9 – 51 30 – 42 30 – 51 42 - 51

Me

an

dif

fere

nce

Temperatuer pair/ °C

Graph of mean difference above critical value

Mean difference

Critical value

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10.0 Bibliography

[1] "Escherichia Coli." Medical News Today. MNT, 08 June 2011. Web. 1 Jan 2012.

<http://www.medicalnewstoday.com/articles/68511.php>.

[2] "Gram-negative." Howard Hughes Medical Institute. HHMI, 2011. Web. 1 Jan 2012.

<http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_2.html>.

[3] "Transformation." Science & Technology Education Program. Lawrence Livermore National

Laboratory, n.d. Web. 1 Jan 2012. <http://education.llnl.gov/bep/science/10/tLect.html>.

[4] Rapoza, Maria. Transformations. Burlington: Carolina Biological Supply Company,

[5] Zimmer, Marc. Green Fluorescent Protein. N.p., 15 June 2011. Web. 1 Jan 2012.

<http://gfp.conncoll.edu/>.

[6] Gleiberman, Anatoli. "Expression of GFP in adult liver." . National Library of Medicine, 2009. Web. 1

Jan 2012.

[7] "Analysis of Variance Between Groups." Tools for Science. MailTo, n.d. Web. 16 Jan 2012.

<http://www.physics.csbsju.edu/stats/anova.html>.