International Baccalaureate Diploma Program Extended Essay

Biology Determining the effect of changing Calcium Chloride concentration on the transformation

efficiency using the heat shock protocol on Escherichia Coli DH5α strain.

Candidate Name: Ji Ho Rhim

Candidate Number: 2213-081

Word Count: 3667

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Acknowledgements: For help with this investigation and making this possible I would like to acknowledge the following people…

Mr. Lawrence Mr. Johnson

Taejon Christian International School and

Professor Lee of Hannam University

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Abstract

Transformation is the genetic variation of a cell that results from uptake of other DNA. It is known that

in the Ca2+

ions provided by Calcium Chloride, is crucial in heat shock transformation process, one of

the ways scientists can induce transformation.

Thus, this investigation concentrates on determining the effect of changing Calcium Chloride

concentration on the transformation efficiency using the heat shock protocol on Escherichia Coli DH5α

strain.

This investigation tested different Calcium Chloride concentrations at 0.1M, 0.08M, 0.06M, 0.04M, and

0.02M and its effects on transformation efficiency of transformation through heat shock procedures.

The bacteria that was used for this investigation was the standard Escherichia coli DH5α strain which

was exposed to plasmid pUC18, a plasmid with antibiotic resistance. Any CFU formed on an LB

Ampicillin plate were considered as transformed. The transformation efficiency was calculated after

counting the bacteria in CFU after 24 hours of incubation.

The results from the research reveal transformation efficiency that as the concentration of calcium

chloride increased the transformation efficiency also increased. The investigation revealed that at

0.02M and 0.04M washed E. Coli DH5α put through the heat shock procedure had the same average

transformation efficiency, and 0.06M and 0.08M showed a trend of transformation efficiency

increasing as calcium chloride concentration increased. However, 0.01M showed a decrease in

transformation efficiency. Statistical results showed that the transformation efficiency of E. Coli DH5α

was significantly higher for 0.08M than for 0.02M or 0.04M.In addition, although there was a decrease

from 0.08M to 0.1M, statistical results showed that there was no significance difference; thus the

implications of an optimum calcium chloride concentration at 0.08M could be inconclusive.

With the given results of significant data, the conclusion that could be derived was that the

transformation efficiency increases as calcium chloride concentration is increased.

[Word Count: 297]

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Table of Contents:

Acknowledgements………………………………………………………………………….….……….….………………….2 Abstract………………………………………………………………………………………….….……….….…………….……..3 Table of Contents…………………………………………………………………………….….……….….…………………..4 1 Introduction

1.1 Rationale………………………………………………………………………………………………....…………..5 1.2 Transformation

1.2.1 E. Coli DH5α……………………………………………………………………………………..……………6 1.2.2 History of

Transformation…………………………………………………………....……………………….…….…7 1.2.3 Uses of Transformation…………………………………………………………..…..……….………..8

2 Hypothesis…………………………………………………………………………………………………….…….……..…9 3 Variables

3.1 Independent Variables……………………………………….………………………………….…….….….……….……….…11

3.2 Dependent Variables……………………………………………………………………………….….……….….……………12

3.3 Controlled Variables…………………………………………………………………………….……………..13 4 Methodology

4.1 Preparation of Ampicillin-LB agar plates……………………….…………….……………………...14 4.2 E. Coli Stock

Preparation………………………...…………….……………………………………….….….….….….….…14 4.3 Heat Shock Transformation Procedure…..…………...………………...…………………….…….15

5 Data Collection 5.1 Raw Data Collection

5.1.1 Qualitative Data……………………………………………………………………………….……….…17 5.1.2 Quantitative Data…………………………………………………………………………………..…...23

5.2 Data Processing 5.2.1 Calculation of Transformation

Efficiency……………………………………………………………………………………………………..24 6 Statistical analysis

6.1 ANOVA Test…………………………………………………………………….…..…..….…..……………………………..27

6.2 Tukey’s HSD Test………………………………………………………………………………………………………..…..…......27

7 Evaluation 7.1 Explanation……………………………………………………………………………………………….…..……29 7.2 Uncertainties and

Limitations…………………………………………………………………………………………………….…...30 7.3 Way to Improve……………………………………………………………………………..…..…...….….….31 7.4 Further investigations…………………………………………………….…………...……..…….…..…..33

8 Conclusion………………………………………………………………..….………………….………………..….......34 9 Bibliography………….…..….………………….….….….…………..………...….….….…………….……….......35 10 Appendix..….……….………….….….….….………….….…..….…………………………………………………….36

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1.0 Introduction 1.1 Rationale

The prokaryotic bacterium has long been accepted as the most basic and resilient

single organism in the world. In understanding life, understanding the bacteria is

crucial. Among the more recent discoveries about the bacteria, the idea of

transformation has been shown to be one of the bacteria’s most important ability.

Thus I found, transformation to be a worthy area of research. During transformation,

an important consideration is in which methodology to induce competency1 to the

bacteria cells.

There are two methodologies in inducing competency of bacteria cells - the

electroporation method2 and the heat shock method3. In schools, because of the

expensive machines required for the electroporation method, the heat shock

method is widely adopted. However, the heat shock method is not an inexpensive

protocol as well. Thus, it is important to increase the efficiency of such process as

much as possible. A variable that can be changed in order to increase such efficiency

is Calcium Chloride.

Thus, by determining the optimum Calcium Chloride concentration for the Heat

Shock methodology, I believe I will help in contributing to the furthering of bacterial

transformation research to any fellow students who will engage in similar

experiment about transformation

Therefore my research topic is…

Determining the effect of changing Calcium Chloride concentration on the

transformation efficiency using the heat shock protocol on Escherichia Coli DH5α

strain.

1 Being able to uptake new genetic information 2 See Appendix B

3 Will be further discussed later on in the investigation

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1.2 Transformation

1.2.1 E. Coli DH5α

E. Coli DH5α is a rod-shaped gram negative bacterial strain and knowing this is

important in the process of transformation, as it is the gram negative structure that

allows the transformation process to work with the variable of calcium chloride.

Figure 1.) Difference between Gram Positive and Gram Negative Bacteria Structure

Gram-negative bacteria[1] also have no nuclear membrane and the chromosome is a

large circular duplex with a membrane attachment site and a single origin of

replication.

This explains why E. Coli DH5α highly transformable; in addition to being recA-4

which makes E. Coli DH5α to be very popular for transformation protocols. In

addition, E. Coli DH5α is a relatively harmless strain of gram-positive bacteria, thus

can be used safely in a lab, although contamination precautions should always be

taken.

4 RecA is a protein that allows repair of genetic material, RecA- means it is deficient of this protein

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1.2.2 The History of Transformation The history of transformation began with the Griffith Experiment in 1928 [2]. This

experiment led to the discovery that non-virulent5 bacteria are able to uptake

genetic information of virulent6 bacteria. In turn, it paved the way for the Avery-

MacLeod-McCarty Experiment in 1944, which showed that isolating DNA from a

virulent strain could be made into a non-virulent strain.

This phenomenon of uptake and incorporation of foreign DNA became known as

transformation. Through further investigation, scientists discovered that these

foreign DNA that were taken in by bacteria were not chromosomal DNA that were

imagined earlier, but circular pieces of DNA called plasmids.

Investigation into the plasmid has led to new discovery that plasmids do not carry

essential cellular genetic codes. However, the plasmids do carry beneficial DNA

which explains how the non-virulent Streptococcus pneumoniae7 had become

virulent and vice-versa that was shown through the first transformation experiments.

Figure 2.) Griffith Experiment and its Results

5 Harmless 6 Harmful

7 Virulent strain of bacteria

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1.2.3 The Uses of Transformation

The uses of transformation can range from creating vaccines to glowing bacteria. By

manipulating the DNA structure of virulent bacterium, such as in Streptococcus

pneumoniae as Frederick Griffith did in his experiment, scientists can create future

vaccines as changing the DNA of the virus to a milder form and improve immunity

towards particular viral problems.

Other notable medical breakthrough as results of transformation was the use of

human insulin harvested from transformed bacteria. This allowed patients suffering

from diabetes to have a ready supply of insulin and avoid allergic reactions towards

introduced insulin as it was taken from humans, not sheep or cattle.

The usage of transformation to introduce Green Fluorescence Protein or GFP in an

organism proved the potential of transformation in 2008 as it was awarded the

Chemistry Nobel Prize[3]. This GFP, found in jellyfish, creates bioluminescence, and

almost immediately glows when synthesized. Thus GFP would have the potential to

perform non-invasive diagnosis and protein tracing, and act as a marker for

researchers in a diagnostics test of a certain patient.

Figure 3.)A fusion protein comprising the RR-signal peptide of trimethylamine N-oxide (TMAO) reductase

linked to green fluorescent protein (GFP) was expressed in wild-type E. coli cells (top panel) or cells lacking a functional tatC gene (lower panel).

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2.0 Hypothesis Today it is widely accepted that most bacteria are incompetent. Thus, in order to

make these bacteria competent this experiment will induce competency through

heat-shocking procedures[4]. The method works as follows.

First, incompetent bacteria are subjected to heat shock which then activates HSP8.

This then causes the pores on the membrane of the bacteria to either dilate or form

new pores. Then as the Calcium Chloride is introduced, it provides Ca2+ 9. Bacterial

cells will become competent. The below figure shows how competency induced

through the use of Calcium Chloride

Figure 4.) Calcium Chloride inducing Competency in Gram Positive Bacteria

One of the most important steps given above is the introducing of calcium chloride,

as it will be the factor allowing admittance of the plasmid vector10 to enter to the

bacteria or artificial competency in the bacteria. If the concentration of calcium

chloride will increase so will the numbers of the Ca2+, which will thus induce more

entrance of plasmids. In turn, it will allow greater transformation efficiency as it

increases the number of transformed bacterial CFUs with the same amount of

plasmid introduced during the experiment.

8 Heat Shock Proteins, or proteins that are expressed when the bacterial cell is subjected to sudden increase in temperature 9 A Divalent Cation that permits the entrance of plasmids through the hydrophilic cell membrane layer.

10 A vehicle used to transfer foreign genetic material into another cell

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It is crucial in locating the effects of decreasing or increase the concentration of

calcium chloride as it will allow the test to not waste any amounts of plasmid and

provide improvements to the Heat Shock method protocol. However, because the

exact effects that calcium chloride will actually have on the cell, it is difficult to say if

there is something as too high of a concentration of calcium chloride, thus it may be

the case there is limiting efficiency in Calcium Chloride. Most of the exact mechanics

of transformation is unknown and the hypothesis is all theoretical although widely

accepted.

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3.0 Variables 3.1 Independent Variables

Washing bacterial cells with calcium chloride is one of the major factors in

determining the amount of transformed bacterial cells created, which will be

counted by CFUs11. By introduced divalent cation Ca2+, the uptake of plasmids

becomes possible as plasma membrane begins to intake the cation with the plasmid.

In the following diagram, the calcium chloride concentrations that will be used in the

investigation will be given.

Calcium Chloride Concentration / M Expected Observations

0.10 Second most CFU is represented

0.08 Most CFU is represented

0.06 Third most CFU is represented

0.04 Second least CFU is represented

0.02 Least CFU is represented

Table 1 – Range of Calcium Chloride Concentration

To make the different concentrations of calcium chloride, a stock solution of 0.1M

calcium chloride will be used, and the rest of the concentrations will be created

through dilution of this 0.1M solution stock using a graduated cylinder.

Concentration of CaCl2 produced / M Amount of 0.10M CaCl2 / mL Amount of Water / mL

0.10 100 0

0.08 80 20

0.06 60 40

0.04 40 60

0.02 20 80

Table 2 – Dilution of the 0.10M solution to create lower concentrations

11 Colony Forming Units

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3.2 Dependent Variable: The Transformation Efficiency in Amount of CFU’s formed in the experiment. The number of CFUs produced at the end of the experiment is decided by the

transformation efficiency[5] of the experiment. Bacterial colonies that survive the

conditions in the LB Ampicillin plate will be counted in CFUs, and the transformation

efficiency will be calculated as follows:

Step 1.) Finding the total mass of plasmid in fraction

(Mass of plasmid used) x (fraction of suspension put on plate/total volume of suspension)

Step 2.) Calculating the Transformation Efficiency

Transformation Efficiency = Total Number of Colonies / Total Mass of Plasmid in Fraction

e.g.)

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3.3 Constant Variables

Type of Bacteria used:

The bacteria used will be E. Coli DH5α for all trials. This is a standard strain that is

used in transformation experiments, and is known to be non-pathogenic and

relatively safe to harness.

Plasmid used:

pUC18 plasmid 12will be used throughout the experiment and will serve as a marker

for Ampicillin resistance.

Antibody used:

Ampicillin, an anti-biotic in the penicillin family, will be used to screen for the

transformation of the bacteria. Ampicillin will also provide the plates some form of

resistance from other contamination.

Incubation period:

The incubation period of the transformed bacteria will be kept constant at a 24 hour

period. This is to ensure that the change in incubation period does not change the

amount of CFUs to be formed.

Temperature of Incubation:

The temperature of the incubation during the incubation period will be kept at the

optimum temperature 37°C

12 See Appendix C

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4.0 Methodology 4.1 Preparation of Ampicillin-LB agar plates

1. 1 L of LB agar 13is prepared

2. At 56°C, 0.200 mg of Ampicillin is added to the LB agar.

3. Mixture is swirled thoroughly

4. Pour in petri dishes

5. The dish is turned upside down after 1 minute

6. Store in refrigerator

Figure 5.) LB Plate Preparation

4.2 E. Coli Stock Preparation

1. 200 μl of LB Broth 14is put into a microcentrifuge tube

2. Using a sterile loop, 10 CFUs of E. Coli are put into the LB Broth.

3. Microcentrifuge tube is stirred using vortex.

4. Microcentrifuge tube is labeled and left to incubate for 24hrs

13 See Appendix A for preparation method

14 See Appendix A for preparation method

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4.3 Heat Shock Transformation Procedure

Duplicate experiment

Remove Petri dishes from incubator and count CFUs on a black background

Place the plates upside-down inside an incubator at 37 C and incubate for a day

Take 100μl of each concentration three times and swab it accordingly with sterile cotton swabs to the three individually labeled LB ampicillin plate

Add 250μl of LB Broth into each of the centrifuges and vortex each of the centrifuges for one minutes

Incubate in ice for 10 minutes

Heat shock in 42 C water bath for 90 seconds

Incubate in ice for 15 minutes

Vortex the micro centrifuge tubes

Add 5μl of the plasmid into the microcentrifuge

Place in ice bath for 15 minutes

Label microcentrifuge tubes

Take the ice-cold CaCl of different concentrations and put 150μl in each of the different concentration into 5 different autocalved microcentrifuges

Using a 50μl micropipette place 15μl of Bracteria-LB broth solution into each of the five microcentrifuges

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Heat Shock Transformation Procedure

Diagram 1.) Heat Shock Transformation Procedure Example

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5.0 Data Collection 5.1 Raw Data Collection

5.1.1 Qualitative Data

Figure 7.) Positive Control Plate – To check if bacteria survive transformation protocol

Figure 6.) E. Coli Stock Plate – To check if bacteria is viable

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[Left] On this plate it possible to observe the production of 2 CFUs. It is notable to make sure not mistake the contaminants with the CFUs. Contaminants tend to be much more wider in radius than a bacterial colony. [Right] On this plate it possible to observe the production of 5 CFUs. It is notable to see that there is no contaminants and that compared to the 0.02M CaCl2 washed transformation it has more CFUs.

Figure 8.) Trial 1 CFU count of 0.02M (left) and 0.1M (right) CaCl2 washed E. Coli DH5α

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[Left] On this plate it possible to observe the production of 4 CFUs. There are no observable contaminants present on this plate. [Right] On this plate it possible to observe the production of5 CFUs. There are no observable contaminants present on the plate.

Figure 9.) Trial 1 CFU count of 0.04M (left) and 0.08M (right) CaCl2 washed E. Coli DH5α

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[Left] On this plate it is possible to count three CFUs. Many large contaminants are observable and may have restricted visible CFU growth that may have grown within the parameters of the contaminants [Right] On this plate it is possible to count 7CFUs, there are some contaminants present. Compared to the first trial, there is a great amount of CFUs present, which again show that Trial 1 CFU growth may have been restricted by the contaminants

Figure 10.) Trial 1 (left) and Trial 2 (right) CFU count of 0.06M CaCl2 washed E. Coli DH5α

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[Left] On this plate it is possible to observe the production of 3 CFUs, which is similar to the number found in the first trial, showing some amount of consistency between the two trials. [Right] On this plate it possible to observe the production of 3 CFUs. It is notable to make sure not mistake the contaminants with the CFUs. Contaminants tend to be much more wider in radius than a bacterial colony. It is also notable that there are many more contaminants than other plates, thus when counting the CFUs more precautions were taken. The less amount of CFUs compared to the first trial may have been due to the contaminants

Figure 11.) Trial 2 CFU count of 0.02M and 0.10M CaCl2 washed E. Coli DH5α

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[Left] On this plate it is possible to observe the production of 2 CFUs, and small contaminants found sparsely on the plate, although difficult to see in the picture the difference, on the real plate, it is clear that the two splotches are contaminants. [Right] On this plate it is possible to observe 6 CFUs, the top-most are two CFUs very close together, in the case of further incubation, it is notable the two CFUs may have combined into one.

Figure 12.) Trial 2 CFU count of 0.04M (left) and 0.08M (right) washed CaCl2 washed E.

Coli DH5α

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5.1.2 Quantitative Data

Table 3.) Table of Countable CFUs in each plate

Counted CFUs for each Trial and Concentration

Concentration of CaCl2, mol dm-3 Trial 1 Trial 2 Average Number of CFUs counted

0.10 5 3 4

0.08 5 6 5.5

0.06 3 7 5

0.04 5 2 3.5

0.02 2 3 3.5

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5.2 Data Processing

5.2.1 Calculation of Transformation Efficiency

In order to calculate Transformation Efficiency, the total mass of plasmid used must

be identified. The formula below can be used to find this…

Total Mass of plasmid = (Total mass of plasmid used) x

Mass of Plasmid used = (Concentration of Plasmid) / (Volume of Plasmid Solution

Used)

= (50 ng/μl) x (5 μl)

= 250 ng

= .25 μg

Total volume of Suspension = 600 μl

Fraction of Suspension put on Plate = 100 μl

Therefore, the Total Mass of Plasmid in Fraction = 0.25 x

= 4.17 * 10-2

Now the transformation efficiency can be calculated as

Transformation Efficiency =

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Table 4.) Transformation Efficiencies for Trials 1 and 2 at different Concentrations of Calcium Chloride Solution

Concentration of CaCl2 Solution, mol dm-3

Trial 1 Trial 2 Total Average CFUs formed Transformation Efficiency,

(colonies / μg)

0.10 5 3 8

= 191.85

0.08 5 6 11

= 263.79

0.06 3 7 10

= 239.81

0.04 5 2 7

= 167.87

0.02 2 5 7

= 167.87

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Graph 1.) Calcium Chloride Concentrations against Transformation Efficiency

0

2

4

6

8

10

12

0.02 0.04 0.06 0.08 0.1

Tra

nsf

orm

ation E

ffic

iency

/ co

lonie

s μg

-1

Calcium Chloride Concentration/ mol dm-3

Calcium Chloride Concentration/ mol dm-3 against Transformation Efficency/

colonies μg-1

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6.0 Statistical Analysis 6.1 ANOVA TEST

In order to further analyze the data for significance, the ANOVA (Analysis of Variance Test) will be preformed to compare the different concentrations of calcium chloride using the raw data collected. Through ANOVA, it will be possible to see whether the Null or Alternate hypothesis should be accepted Null Hypothesis (H0) – No significant difference among different concentrations

Alternate Hypothesis (HA) – There is significance among different concentrations.

Table 5.) Results of the ANOVA test

11.7 (F Ratio) > 4.5 (F Critical) – Null Hypothesis is rejected and Alternate Hypothesis is accepted. Further tests will be preformed to investigate which groups are significant

6.2 Tukey’s HSD Test Tukey’s HSD (honestly significant difference) test compares all possible pairs of groups to see which group pair is greater than the critical value. The critical value of the Tukey’s HSD is found to be as follows…

Critical Value = q(α,k,N-k)

α = 0.05 (5% is set at significant level) k = 5 (total number of groups) N-k = 5 (total number of results – total number of groups)

Therefore q-value is 1.47% and critical value =

= 1.68

ANOVA

Source of Variation Sum of Squares df Mean Squares F Ratio P-Value F Critical

Between Groups 60.8 2.0 30.4 11.7 Comp.

Generated 4.5

Within Groups 20.7 8.0 2.6

Total 25.2 12.0

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Calcium Chloride Concentration, mol dm-3

Mean Difference Critical Value

0.02-0.10 4 – 3.5 = 0.5 1.68

0.02-0.08 5.5 - 3.5 = 2 1.68

0.02-0.06 5.0 - 3.5 = 1.5 1.68

0.02-0.04 3.5 - 3.5 = 0 1.68

0.04-0.10 4 - 3 = 1 1.68

0.04-0.08 5.5 - 3.5 = 2 1.68

0.04-0.06 5 - 3.5 = 1.5 1.68

0.06-0.10 5 - 4 = 1 1.68

0.06-0.08 5.5 - 5 = 0.5 1.68

Table 6.) Comparison of mean difference to critical value Two pairs have mean differences that are greater than the critical value: 0.02 – 0.08 M calcium chloride concentration 0.04 – 0.08 M calcium chloride concentration

Notably, both include the concentration of 0.08M calcium chloride concentration.

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7.0 Evaluation 7.1 Explanation

From the results given from the experimentation, it could be seen that as the

concentration increased from 0.02M to 0.08M, the transformation efficiency

increased as well. At concentrations of 0.02M and 0.04M, the transformation

efficiency remained constant, and efficiency slightly decreased at the concentration

of 0.1M Calcium Chloride.

The reasons for the data being represented as it is could lie in the following reasons:

1.) At 0.02M and 0.04M the concentrations of Calcium Chloride solution is too low

to provide any significant change in transformation efficiency.

2.) At 0.06M and 0.08M the concentrations of Calcium Chloride show a greater

transformation efficiency. At these concentrations, the Ca2+ ions are able to

greatly affect the cell membrane and help the plasmid to bind to the membrane

to an extent that transformation efficiency increases significantly.

3.) At 0.1M, the concentration is too high and although more Ca2+ ions were present,

it would not help in increasing the rate or amount of binding of the plasmid to

the membrane.

However the fact that 0.02M and 0.04M concentrations of Calcium Chloride does

seem irregular in the fact that even though there is growth shown for a

concentration as low as 0.02M; yet there is no difference with a calcium chloride

solution with doubled concentration. This may have had to do with random and

systematic error that will be further discussed in uncertainty and limitation.

In addition it is important to note that because there is no significant difference

between 0.1M and 0.08M; 0.1M is likely to be an inconclusive piece of data. However,

because there is a significant difference between 0.02M and 0.08M as well as 0.04M

and 0.08M, it is conclusive that there is a trend that as calcium chloride

concentration increases, the transformation efficiency does increase.

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7.2 Uncertainty and Limitation

Many other uncertainty and limitations can be found asides from the theoretical

aspects of this investigation.

One of these errors could occur while preparing a stock of E. Coli DH5α suspended in

L.B. Broth. Since bacteria have a natural tendency of clumping, it could be said that

either not all the bacteria that was provided in the stock came with the same amount

of bacteria every time the 15μl of bacterial solution was used for each plate.

Although there were countermeasures taken to try to prevent this error from having

significant effects on the results, such as using the vortex before the bacterial

solution was taken out for use, the effects of clumping may still have played a part in

producing the given results

In addition, contamination could have introduced a new strand of bacteria that is

resilient to the antibiotic used in the experiment. Other contaminants such as fungi

spores could also be introduced into the plate causing the fungi to release a toxin

that kills bacterial CFUs. In some of the qualitative data shown earlier, it shows the

likelihood that contaminants may have debilitated CFU growth.

In addition, the cotton swabs used in the plating after the heat shock may have

absorbed potential CFUs severely reduced the numbers that would have represented.

A notable limitation while performing the experiment could be little number of trials

preformed, which would have thus led to a failure to collect data that may be

significantly different according to ANOVA and the Tukey’s HSD test. Due to a

shortage of plasmid puc18, the trial was limited to duplication rather than the

preferred triplicate. In addition the storage of the puc18 was insufficient for the

experiment. The optimum temperature of storage of plasmid is -20°C whereas the

school fridge would only go down to -4°C. Thus, plasmid degradation over time may

have led to a low number of CFUs that were transformed.

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7.3 Ways to Improve

The most important factor to improving this investigation would be to increase the

number of trials preformed for each concentrations of Calcium Chloride. For two

trials would hardly pass as sufficient in making any of the data viable, by increasing

the number of trials, the reliability of the data as well as accuracy and thus credence

will be furthered.

In addition, a more in-depth range of Calcium Chloride could be used, while using

0.075M as a control concentration for Calcium Chloride concentration instead of

0.1M. Because there is a trend that is difficult to predict between what happens in

the 0.02M and 0.04M concentration area as well as a sudden decrease of

transformation efficiency in 0.1M could be then observed with an increased number

of trials and more in-depth range to see a clearer trend to what is happening.

Having more effective equipment such as a sonicator or a rotating incubator to

prevent the bacteria from clumping would help in the preparation of the bacterial

stock within the L.B. Broth to make sure that equal amounts of bacteria go into each

respective microcentrifuge tube; thus controlling the number of bacteria that goes

into with greater efficiency.

The greater planning of experiment would also improve the experiment greatly. The

experiment took a much greater time than it was first intended to due to

complications with faulty material and shortages of many things such as

micropipette tips and sterile cotton swabs. This led to the contribution of the

plasmid degradation that occurred throughout the experiment and also planning a

methodology to sustain the plasmid in a better environment may have brought a

better outcome for the experiment results.

Lastly, an electrical bacterium counter would help greatly into identifying the

bacterial CFUs because the human eye works only so well. Smaller colonies will be

32

overlooked with the naked human eye as well as colonies being mistaken as air

bubbles. Having an electrical bacterium counter will help to reduce the human error

in the final steps of the experiment.

Thus, in these ways many limitations and uncertainties can be removed.

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7.4 Further Investigation

The effects of bacterial transformation hold great potential in the future of

microbiology as well as the medical world. GFP is a leading application of this as GFP

can not only be applied to bacteria but also the human eukaryotic cells as well. The

prime example being the human liver which has shown through a methodology

called transfection. GFP is only just one of the many applications of possibly many to

come in the future. Research about the human immune system currently is

beginning to apply the use of such transformation on the idea of how scientists

could possibly use this sort of method to implant genetic information to combat

certain illness.

To a student who is furthering his or her understanding of transformation,

investigations of bacterial transformation could be down by changing the

independent variable about the study. In this investigation, the independent

variable was the Calcium Chloride. Other independent variables that could be

changed to further one’s knowledge of transformation could be. Applying different

providers of the cation ions necessary for the transformation such as Manganese

and Potassium which may have a greater effect due to its polarity could be

effectively ways of expanding the investigation of transformation. In addition, other

variables from other steps of the transformation protocol could be changed. Such

variables include the temperature of the heat shock to induce the HSP to work or

even the type of plasmid which would give the highest transformation efficiency.

In addition, the opposite could be studied into seeing what would reduce the

transformation efficiency of a bacterial strain. In the world today, many virus and

bacterial strains are achieving levels of immunity towards many of Man’s antibiotics.

An example of this can be seen in the abysmal effects of penicillin on normal

bacterial strains such as the E. Coli DH5α. To prevent the spread of epidemics, a

study of how to reduce the bacteria’s ability to adapt and take in the new genetic

information through reverse engineering transformation would also be an

34

interesting route of furthering the investigation of transformation.

Lastly, other methods of transformations could also be tried. In this investigation,

the heat-shock method was applied. However, the heat-shock method is not known

to be the most efficient method for bacterial transformation. Alternative

transformation methods such as electroporation could be studied given the right

equipment and materials. However, this method does have its downfalls for it may

be too expensive for one to use.

8.0 Conclusion

With the evidence that has been obtained by the investigation, when using the heat-

shock method of transforming bacteria, although finding an the optimum

concentration of Calcium Chloride solution may be inconclusive, it is clear to see that

as calcium chloride concentration increases from 0.02M to 0.08M, the

transformation efficiency also increases.

With further investigation with tests in the range of the concentrations between

0.06M and 0.08M may reveal that there exist precise optimum concentrations of

calcium chloride. However, concentrations that are lower may not be providing the

calcium ions with the greatest efficiency while concentrations greater than the range

may be producing too much calcium ions that the membrane becomes degenerated.

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9.0 Bibliography

[1] Salton MJR, Kim KS 1996, Structure in: Baron's Medical Microbiology, 4th edn, Austin, Texas.

[2] WARD’S Natural Science Establishment 2002, Glowing Bacteria: Transformation with a Firefly Gene Lab Activity Student Study Guide, Rochester, NY.

[3] Nobelprize.org 2008, The Nobel Prize in Chemistry – Press Release, Nobel Prize Press Release, viewed October 3rd 2010, <http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/press.html> [4] Iowa State University Office of Biotechnology 2003, DNA TRANSFORMATION OF BACTERIA – AMPICILLIN RESISTANCE, Ames, Iowa. [5] EDVOTEK 2005, Transformation of E. coli with pGALTM (blue colony), Bethesda, MD

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10.0 APPENDIX APPENDIX A – LB Agar Preparation

Materials Required:

2.5 g – Yeast Extract 5 g – Sodium chloride 5 g – Tryptone 7.5 g – Bacteriological Agar 500mL - Water

Methodology:

1. Measure the Yeast Extract, Sodium Chloride, Tryptone, Bacteriological Agar to 2.5g, 5g,

5g, 7.5g respectively using the electronic balance and spatula and into the 250mL

beaker

2. Put the reagents into the 1L conical flask

3. Using 500mL of water that is measured using a volumetric flask, use the spatula and

remove all remaining reagents in the beaker into the conical flask.

4. Cover the conical flask’s mouth with the 250mL beaker and place inside the autoclave.

5. Autoclave until 17psi and then turn the autoclave off, leaving it to remain inside the

autoclave for 15minutes.

6. Remove the conical flask from the autoclave and begin to swirl gently to not arouse

any bubbles but enough to stop solidifying.

Figure 13.) preparation of LB Agar

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APPENDIX B –ELECTROPORATION METHOD

Diagram 2.) Electroporation

Unlike the Heat Shock Method, which uses the heat shock to temporarily allow

the cell membrane to be permeable, in electroporation electrical shock is

incorporated to create temporary competency within the cell.

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APPENDIX C – pUC18

Diagram 3.) pUC18 Vector

<Image Taken from http://www.genscript.com/vector/SD1162-pUC18_plasmid_DNA.html>