Plasmid transformation using pUC18 on E Coli using heat shock method

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International Baccalaureate Diploma Programme Extended Essay

BIOLOGY Determining the optimum temperature that produces the highest transformation efficiency rate using the heat shock transformation method on modified Escherichia coli strain.

By JORREL TOO YU-XANN

Candidate Number : CQV920 (002206-017) Word Count : 3,981

Jorrel Too Yu-Xann

CQV920(002206-017)

Abstract

Transformation is a very powerful tool in expanding the boundaries of medicine and life sciences. It simply means inserting extracellular DNA into an organism (usually bacteria) using a vector, thereby transforming it. The most common vector used is plasmid, selfreplicating circular strands of DNA that can be modified. Once the vector has been absorbed by the organism, certain proteins not normally synthesized will be present. These proteins may confer antibiotic resistance while others may secrete useful proteins such as insulin to be harvested. However, most bacteria are unable to absorb plasmids naturally. The heat shock method artificially induces competence using a sudden surge in temperature briefly. This research therefore attempts to locate which temperature will allow for optimum transformation efficiency. This research will test a range of temperatures which range from 0.0°C (control – no heat shock), 25.0°C (room temperature), 37.0°C, 42.0°C to 60.0°C. The bacterium of choice is a modified Escherichia coli strain which allows it uptake of the plasmid pUC18. pUC18 is a 2686 base pair 1 length vector that grants the bacteria resistance against ampi cillin. Thus, any bacteria that is present on an LB:Amp 2 agar plate is said to be transformed. Untransformed bacteria would not be able to grow on the lethal medium. The transformation efficiency will be calculated based on the number of colonies present after incubation overnight. Results reveal that 42.0°C has the highest transformation efficiency followed by 37.0°C. Further analysis using ANOVA and Tukey’s HSD test shows that the temperature of 42.0°C is more significant and therefore more efficient compared to 37.0°C. At both 0.0°C and 60.0°C, transformation efficiency was zero. At 25.0°C, transformation efficiency was insignificant because growth was attributed to error. The study suggests that 42.0°C is the optimum temperature for the heat shock method. (291 WORDS) 1

Information obtained from Fermentas Life Sciences (http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm) 2 Luria broth agar containing ampicillin antibiotics.

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Jorrel Too Yu-Xann

CQV920(002206-017)

Table of Contents Abstract................................................................................................................................1 Contents...............................................................................................................................2 1.0

Introduction.............................................................................................................3

1.1

The Uses of Transformation ..................................................................................4

1.2

Rationale for Study ................................................................................................6

2.0

Hypothesis ...............................................................................................................7

3.0

Variables ..................................................................................................................8

3.1

Manipulated Variable............................................................................................8

3.2

Responding Variable..............................................................................................9

3.3

Constant Variable ................................................................................................10

4.0

Methodology .........................................................................................................11

4.1

Preparation of LB:Amp Plates ............................................................................11

4.2

Preparation of Competent Bacteria ...................................................................11

4.3

Transformation ...................................................................................................12

5.0

Data Collection ......................................................................................................15

5.1

Raw Data............................................................................................................15

5.2

Calculation of Transformation Efficiency ..........................................................16

5.3

ANOVA Test .......................................................................................................19

5.4

Tukey’s HSD Test ...............................................................................................20

6.0

Evaluation ..............................................................................................................22

6.1

Uncertainties and Limitations ...........................................................................24

6.2

Ways to Improve ...............................................................................................25

6.3

Further Investigations .......................................................................................26

7.0

Conclusion..............................................................................................................27

8.0

Appendix................................................................................................................28

9.0

Bibliography...........................................................................................................40

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Jorrel Too Yu-Xann

CQV920(002206-017)

1.0 – Introduction In 1928, Frederick Griffith discovered that a non-virulent strain of Streptococcus pneumoniae would turn virulent when exposed to virulent strains of the same species (1). The picture below demonstrates the early experiment:

In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered that there were special loops containing genetic information that can be transferred from one bacterium to another (1). When the heat caused the cell membrane to rupture, DNA was released from the heat-killed pathogenic strain of Pneumococcus into the surrounding medium. This DNA was then absorbed and incorporated into the live nonpathogenic strain which then expressed the newly acquired genes. This phenomenon was termed transformation.

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Jorrel Too Yu-Xann

CQV920(002206-017)

It was only later discovered that the DNA absorbed was circular pieces of DNA that exist and replicate independently of the chromosomal DNA. Called plasmids, they carried genes for specifying a wide range of functions. However, plasmids differ from chromosomal DNA in the sense that the functions encoded only benefit the cell in restricted surroundings. Furthermore, plasmids do not contain essential cellular genetic codes.

1.1 – The Uses of Transformation My interest in transformation came about the first time I saw a transgenic organism. It was a tobacco plant that glowed yellow due to the presence of the firefly lux gene3. This caught my attention because it was something never seen before. This led me to my first transformation experiment. Wanting to create my own transgenic organism, I purchased a transformation kit 4 to further understand how the mechanics work. After much testing and trials, I finally created E.coli that glowed yellow in the presence of luciferin5.

Glowing E.coli

Figure 1 – Transformed E.coli glowing due to luciferase.

3

See Appendix 1. A kit containing all necessary materials for conducting a transformation experiment. 5 A class of pigments that emit light when used by luciferase or another photoprotein. 4

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Jorrel Too Yu-Xann

CQV920(002206-017)

There are many uses for transformation. One of its earliest uses is human insulin harvested from transformed bacteria. This is especially useful because patients suffering from diabetes now have a ready supply of insulin at hand. As the previous source of insulin was harvested from sheep and cattle, allergies will rarely occur as the insulin now used is human insulin.

The potential of transformation was acknowledged in the 2008 Chemistry Nobel Prize Award. Green Fluorescence Protein (GFP) is another protein that allows for bioluminescence like luciferase (2). However, unlike luciferase, GFP does not require a substrate and thus immediately glows once it has been synthesized. This allows it to perform certain roles like non-invasive diagnosis and protein tracing. By attaching GFP to a DNA sequence, GFP will be synthesized along with the protein allowing researchers a marker to look for.

Figure 2 – Mouse with brain tumor expressing DsRed6

6

A red fluorescent protein (emission maximum at 583 nm) that, like GFP, can be genetically conjugated to proteins to make them fluorescent. First isolated from tropical corals of the Discoso ma genus.

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Jorrel Too Yu-Xann

CQV920(002206-017)

1.2 – Rationale for this study Although there is much emphasis on transformation, not many IB students are able to experiment on transformation. The high costs of plasmids, the fast degradation time, and low transformation rate make transformation experiments highly risky. In order to encourage others to explore this field, I want to increase the transformation rate by

determining the optimal temperature for heat shocking. Heat shocking is vital in determining the amount of bacteria transformed because the bacteria need to be made artificially competent in order to absorb the plasmid. The heat shock method has several variations and this is what has intrigued me to conduct a study on the method itself. The temperatures of 37°C and 42°C are both common temperatures found in scientific sources worldwide in terms of heat shock (3). However, which temperature works best for the IB student who has both limited number of trials and limited time?

By determining the optimum temperature for heat shocking, fellow IB students will be more confident in conducting transformation experiments due to greater certainty of results

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.

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Jorrel Too Yu-Xann

CQV920(002206-017)

2.0 – Hypothesis7 Most bacteria are unable to absorb plasmids from their surroundings. Hence, these bacterial species are said to be incompetent. The purpose of heat shocking bacteria is to induce competency in the bacteria. This method is theorized to work as follows:-

1. Incompetent bacteria are subjected to a sudden increase in temperature. 2. Heat Shock Proteins (HSPs) are activated. 3. Pores found among the bacteria cell wall increase in size / new pores created. 4. Calcium (Ca2+) ions neutralize the negative charges found on both the plasmid DNA and the phospholipid bilayer. 5. Chloride (Cl-) ions enter bacteria. Sudden influx of chloride ions causes shift in osmotic concentration of bacteria. 6. Water (containing plasmids) enter the cell. 7. Bacteria are then cooled back to previous temperature. The most important step above is to ensure that the Heat Shock Proteins (HSPs) are activated because the Heat Shock Proteins will activate a cascade reaction which will ultimately lead to artificial competency in the bacteria. With a sudden increase in temperature, certain genes are expressed wherein pores in the plasma membrane form. These pores allow the plasmids to be absorbed into the cell. It is imperative that the optimum temperature be found 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. If the temperature is too high, the bacteria will die due to protein denaturization.

Note: The exact mechanics of transformation is relatively unknown, thus this hypothesis is purely theoretical but widely accepted.

7

See bibliography (4) for a complete list of references quoted.

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Jorrel Too Yu-Xann

CQV920(002206-017)

3.0 – Variables 3.1 – Manipulated Variable: Temperature used for heat shock treatment. The heat shock treatment determines the amount of transformed bacterial cells created. Only when the bacteria are subjected to a sudden increase in heat that the pores in the plasma membrane open, allowing the plasmids outside to be absorbed.

In the following table, is the desired range of temperature which I will use to heat shock the bacteria: Expected Observations

Temperature, (± 0.5°C) 0.0

At 0.0°C, no transformation should occur because heat shock proteins not activated. This temperature serves as a control.

25.0

At 25.0°C (room temperature), very slight chance of transformation occurring due to a change in temperature.

37.0

At both 37.0°C and 42.0°C, I predict that a majority of bacteria

42.0

will absorb the plasmid from the surrounding medium. Further analysis will have to be done to confirm which temperature grants the highest transformation rate.

60.0

No CFU 8 formation at this temperature because at 60.0°C, most proteins would be denatured and thus cellular function ceases. This temperature serves as a control. Table 1 – Range of temperature of heat shocks

To ensure that the temperature is correct, a digital thermometer is used to measure the temperature before transformation takes place.

8

Colony Forming Unit - measure of viable bacteria

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Jorrel Too Yu-Xann

CQV920(002206-017)

3.2 – Responding Variable: The transformation efficiency rate in the amount of CFU’s formed at the end of the experiment. Bacteria will be plated on agar plates containing ampicillin. Ampicillin has antibiotic properties, thus only bacteria which contain the ampicillin resistant gene will be able to survive as they are able to breakdown ampicillin. Untransformed bacteria do not have ampicillin resistance and therefore will not be able to survive on the agar. The surviving bacteria will be counted and transformation efficiency rate will be calculated as follows: Step 1 : Find total mass of plasmid in fraction

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Step 2 : Calculate Transformation Efficiency

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Jorrel Too Yu-Xann

CQV920(002206-017)

3.3 – Constant Variables • Bacteria used will be Escherichia coli. A specially modified strain of E.coli will be used throughout the entire experiment. This

strain can undergo artificial competency, is non pathogenic and relatively safe. Contamination will be prevented using methods such as streaking and control tests.

•

Plasmid used will be pUC189 DNA

pUC18 plasmid contains an ampicillin resistant marker which allows the transformed bacteria to breakdown ampicillin.

•

Ampicillin is the antibiotic of choice.

Ampicillin will be poured into the agar to help screen the transformed bacteria from the untransformed ones. Ampicillin also prevents contamination from external sources.

•

Incubation period will last 24 hours and incubation temperature is at 37.0°C.

This is to ensure that bacterial growth will be systematic throughout the entire experiment. A change in incubation period and/or incubation temperature will result in a change in the bacterial growth rate.

9

See Appendix 2 for full genetic sequence.

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Jorrel Too Yu-Xann

CQV920(002206-017)

4.0 – Methodology 4.1 – Preparation of Ampicillin-LB agar (LB:Amp) plates. 1. 500 ml of LB agar10 is prepared. 2. When LB agar is at a temperature of 57.0°C, 0.300 mg of Ampicillin is added. 3. Mixture is mixed thoroughly and poured into petri dishes. 4. The dish is turned upside-down after 2 minutes and stored in the refrigerator.

4.2 – Preparation of Competent bacteria (Stock). 1. Using a sterilized loop, a single colony of modified E.coli culture is removed and placed into 1.0 ml of ice-cold Calcium Chloride in a 1.5 ml microcentrifuge. Loop is gently tapped to dislodge bacteria. 2. 2.5 µl of plasmid is added to the using a pipette. The microcentrifuge is gently tapped. 3. Microcentrifuge is placed back on ice and bacteria ready to undergo heat shock.

Figure 3 – Ice bath containing competent bacteria 10

See Appendix 3 for recipe and preparation.

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Jorrel Too Yu-Xann

CQV920(002206-017)

4.3 – Transformation 4.3.1 – At temperatures of 37.0°C, 42.0°C, and 60.0°C 1. Waterbaths at temperatures of 37.0°C, 42.0°C, and 60.0°C are prepared. 2. From the microcentrifuge, 100 µl of competent bacteria with plasmid are removed and transferred into a new microcentrifuge (labeled A). 3. Microcentrifuge A is placed in a float and dropped into the 37.0°C waterbath. A stopwatch is started immediately.

Figure 4 – Microcentrifuge, float in water bath.

4. After 2 minutes, microcentrifuge and float is removed and placed back directly on ice for another 5 minutes. 5. After 5 minutes, 75 µl of transformed cells from the microcentrifuge A are removed using a pipette and spread evenly on the surface of a LB:Amp agar plate. 6. Steps 4 to 7 are repeated for temperatures 42.0°C, and 60.0°C. 7. Petri dishes are turned upside down and incubated.

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Jorrel Too Yu-Xann

CQV920(002206-017)

4.3.2 – At Room Temperature (≈25.0°C)

8. 100 µl of competent bacteria from stock is transferred into microcentrifuge B. 9. Microcentrifuge B is placed in a float held by a retort stand. The stopwatch is started immediately. 10. After 2 minutes, the float and microcentrifuge is removed and placed back on ice. 11. After 5 minutes, 75 µl of transformed cells in microcentifudge B is removed using a pipette and spread evenly on the surface of a LB:Amp agar plate. 12. Petri dishes are turned upside down and incubated.

4.3.3 – At 0.0°C 13. 75 µl of competent bacteria from stock is removed using a pipette and spread evenly on the surface of a LB:Amp agar plate. 14. Petri dishes are turned upside down and incubated.

4.3.4 – Collection of results

1. After incubation at 37.0°C for one night, the petri dishes are removed from the incubator. 2. Petri dishes are placed on top of a black cardboard sheet.

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Jorrel Too Yu-Xann

CQV920(002206-017)

Colony Colony

Figure 5 – Inverted Petri dish showing CFU’s

3. Any bacterial colonies are noted. 4. This experiment is duplicated to reduce the experimental error.

4.3.5 – Methods of disposal

1. A strong antibacterial agent is poured onto the surface of the agar. 2. The petri dish is tilted, to wash entire surface of agar, and thrown away in a biohazard waste bag.

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Jorrel Too Yu-Xann

CQV920(002206-017)

5.0 – Data Collection 5.1 – Raw Experimental Data

Temperature, (± 0.5°C)

Trial 1

Trial 2

Average / (CFU)

Plate 1

Plate 2

Plate 3

Plate 4

0.0

–

–

–

–

–

25.0

–

2

–

–

37.0

–

2

2

3

42.0

2

5

2

2

60.0

–

–

–

–

2 = 0.5 4 7 = 1.75 4 11 = 2.75 4 –

Table 2 – Average CFU at different temperatures (–) = No CFU’s found

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Jorrel Too Yu-Xann

CQV920(002206-017)

5.2 – Calculation of Transformation Efficiency Before calculating the transformation efficiency, the total mass of plasmid must first be found. This will be done using the formula shown below:

Total Mass of plasmid = (𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝒎𝒎𝒎𝒎𝒎𝒎𝒎𝒎 𝒐𝒐𝒐𝒐 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝒖𝒖𝒖𝒖𝒖𝒖𝒖𝒖) ×

𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇𝒇 𝒐𝒐𝒐𝒐 𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 𝒑𝒑𝒑𝒑𝒑𝒑 𝒐𝒐𝒐𝒐 𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑𝒑 𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽𝑽 𝒐𝒐𝒐𝒐 𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺𝑺

Mass of plasmid used = (𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝) × (𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣 𝑜𝑜𝑓𝑓 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑢𝑢𝑢𝑢𝑢𝑢𝑢𝑢) = (20𝑛𝑛𝑛𝑛⁄𝜇𝜇𝜇𝜇 ) × (2.5𝜇𝜇𝜇𝜇) = 50𝑛𝑛𝑛𝑛

= 0.05μg

Total volume of suspension = 1000µl

Fraction of suspension put on plate = 75µl

Total Mass of Plasmid in fraction = (0.05) ×

75 1000

= 𝟑𝟑. 𝟕𝟕𝟕𝟕 × 𝟏𝟏𝟏𝟏−𝟑𝟑 𝛍𝛍𝐠𝐠

The transformation efficiency can now be calculated using the formula below:

𝑇𝑇𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸𝐸 =

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝑚𝑚𝑚𝑚𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜 𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 𝑖𝑖𝑖𝑖 𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓𝑓

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Jorrel Too Yu-Xann

CQV920(002206-017)

Temperature (¹ 0.5° C)

Trial 1 (Average CFU’s)

Trial 2 (Average CFU’s)

Total Average CFU’s formed. (Trial 1 + Trial 2)

Transformation efficiency (colonies/ Âľg)

0

–

–

–

25

1

–

1

0 = đ?&#x;Žđ?&#x;Ž. đ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Ž 3.75 Ă— 10−3

37

1

3

4

42

4

2

6

60

–

–

–

1 = đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?. đ?&#x;”đ?&#x;”đ?&#x;”đ?&#x;” 3.75 Ă— 10−3

4 = đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?. đ?&#x;”đ?&#x;”đ?&#x;”đ?&#x;” 3.75 Ă— 10−3 6 = đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?. đ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Ž 3.75 Ă— 10−3

Table 3 – Transformation Efficiencies for Trials 1 and 2 at different temperatures (–) = No CFU’s found

0 = đ?&#x;Žđ?&#x;Ž. đ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Ž 3.75 Ă— 10−3

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Jorrel Too Yu-Xann

CQV920(002206-017)

Temperature/°C against Transformation Efficiency/ colonies µgˉ¹ 1800

T r a n s f o r m a t i o n E f f i c i e n c y

1600

1600

1400

1200 1066.67 1000

800

600

400 266.67 200

0

0 0

0 25

37

42

60

Temperature/ ºC Transformation Efficiency

Graph 1 – Temperature against Transformation Efficiency More tests will be conducted to further analyze the data. Page | 18

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CQV920(002206-017)

5.3 – ANOVA Test

An ANOVA Test (ANalysis Of Variance Test) is used to compare which of the 5 temperatures done in this experiment is significant. For this statistical test, raw data collected (number of colonies formed) is used. The ANOVA test will determine which hypothesis is accept based on the results obtained:

•

Null Hypothesis, H0 = Âľ1 = Âľ2 = Âľ3 = Âľ4 = Âľ5. No significant difference between groups.

The Null hypothesis is selected if the computed F statistic11 is less than the table value12.

If the computed F statistic is greater than the table value, an alternative hypothesis is selected instead.

•

The Alternative hypothesis is defined as H1 = one or more means are different. There is a significant difference between the groups.

Source of

Sum of

Variation

Squares

Between Groups

23.5

Within Group

14.5

Total

38.0

df

Mean Squares

F Ratio

Critical F

P Value

đ?&#x;”đ?&#x;”. đ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Žđ?&#x;Ž

3.06

(computer

(s2) 4

5.875

15

29 30

19

generated)

Table 4 – Results of the ANOVA test

6.078 (F ratio) > 3.06 (F critical) = Null hypothesis rejected and Alternate Hypothesis accepted. 11 12

Named after R.A. Fisher who created it. See Appendix 4.

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One of the groups above has a significant difference from the rest of the group. Further tests, also known as Post Hoc Analyses, will be done to investigate which group is significant.

5.4 – Tukey’s HSD test For this analysis, I will be using Tukey’s HSD (honestly significant difference) test. Tukey’s HSD test works by comparing all possible pairs of groups in an effort to compare which pair is greater than the critical value. Tukey’s HSD test has a critical value13 found to be:������

Critical Value = 4.37ďż˝

��

29

= 4.37ďż˝ 304

= 4.37(0.492)

= đ?&#x;?đ?&#x;?. đ?&#x;?đ?&#x;?đ?&#x;?đ?&#x;?

13

See Appendix 5 for in-depth explanation.

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Temperature , (± 0.5°C)

Mean difference

Critical Value

0.0 – 25.0

0.50 – 0.00 = 0.50

2.15

0.0 – 37.0

1.75 – 0.00 = 1.75

2.15

0.0 – 42.0

2.75 – 0.00 = 2.75

2.15

0.0 – 60.0

0.00 – 0.00 = 0.00

2.15

25.0 – 37.0

1.75 – 0.50 = 1.25

2.15

25.0 – 42.0

2.75 – 0.50 = 2.25

2.15

25.0 – 60.0

0.50 – 0.00 = 0.00

2.15

37.0 – 42.0

2.75 – 1.75 = 1.00

2.15

37.0 – 60.0

1.75 – 0.00 = 1.75

2.15

42.0 – 60.0

2.75 – 0.00 = 2.75

2.15

Table 5 – Comparison of mean difference and critical value14

The following pairs have a mean difference greater than the critical value: •

0.0°C – 42.0°C

•

25.0°C – 42.0°C

•

42.0°C – 60.0°C

Notice that all pairs involve the temperature of 42°C.

14

See Appendix 6 for graphical interpretation.

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6.0 – Evaluation

From the results, transformation efficiency at 0.0°C and 60.0°C is zero. Reasons are:

a) Cells did not take in the plasmid and thus are unable to synthesize beta lactamase which hydrolyzes ampicillin. b) Proteins in the cell have been denatured due to temperature.

At 0.0°C, bacteria did not undergo heat shock. There was no sudden change in temperature (constant at 0.0°C). HSPs were not activated. Thus, bacterial pores are not stimulated and plasmids were unable to enter the cell. Another reason would be that prolonged exposure to cold could have damaged the bacteria thus retarding their growth. E.coli is known to hibernate when exposed to freezing temperatures. Thus, exposed at 0.0°C for an extended period of time could have caused E.coli to being hibernating. When E.coli hibernates, no molecules move in and out of the bacteria. This results in no plasmid uptake and hence, no antibacterial resistance.

At 60.0°C, heat has caused bacterial proteins to be denatured. Bacteria is killed as important enzymes would be unable to maintain their active site and thus can no longer catalyze reactions. HSPs which would otherwise reform the denatured protein have themselves been denatured. Plasmids are also very vulnerable to high temperatures. Subjected to 60.0°C, they rapidly denature. Since both plasmids and bacterial proteins are denatured, transformation is highly improbable resulting in zero transformation efficiency.

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Though transformation efficiency at 25.0°C is low, it might be a result of experimental error. Bacteria transformed at this temperature grew only on one plate (plate 2). Further analysis 15 reveals that this temperature does not reach the critical value. This could be due to contamination from outside sources. Efficiency is low because the temperature increase was not enough to cause HSP activation. HSPs are also termed as stress proteins because they express only when the cell undergoes stress, one example is exposure to high temperature. Since the bacteria experience no stress at 25.0°C, HSPs remained inactivated. Transformation efficiency at 37.0°C can be labeled as average although the mean difference of 37.0°C does not exceed the critical value. Transformation efficiency is average because 37.0°C is a high enough temperature to induce most HSPs to activate but not all. Hence, only a small percentage of HSPs are activated to produce a likewise number of pores. The lower the number of pores, the lower the chance of plasmids entering the bacterium. It could also be due to the length of the transformation. Since heat travels by conduction, a lower temperature would mean slower heat conduction. In other words, at 37.0°C, only the bacteria near the walls of the micro-pipette are transformed. Bacteria in the center of the micro-pipette do not experience an increase in temperature and are not heat shocked.

42.0°C has the highest transformation efficiency. At 42.0°C, all HSPs are activated because the stress bacterial cells undergo is significant enough for the cell to fully respond. Since all HSPs are activated, the most number of pores are created. More pores result in a greater influx of Clions because since the concentration of CaCl is constant, the main difference between each temperature is number of pores created. In other words, two pores will allow 2 ions of Cl- to enter followed by 2 plasmids per unit time (42.0°C) whereas one pore allows only 1 Cl- ion followed by 1 plasmid per unit time (37.0°C).

15

Refer to Tukey’s HSD test, section 5.4.

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Therefore, a brief heat shock is suitable for 42.0°C because pores tend to stay open for the optimal amount of time – long enough for plasmids to enter but short enough to prevent heatdegradation to occur.

6.1 – Uncertainties and Limitations The final optimum temperature cannot be conclusively proven as there is a lack of replicates or trials due to the limited amount of plasmid available. 2 trials on 4 petri dishes are not enough to give a clear conclusion especially regarding transformation which has a high margin of error. Hence, the results collected might not be entirely correct. This experiment used only E.coli. Since heat shock differs from organism to organism, the suggested temperature might not work for other species of bacteria. Furthermore, only a modified strain of E.coli was used. Wild strains might have greater difficulty becoming competent and have a lower transformation rate. The amount of bacteria used for each trial was different because different colonies were chosen from the stock. Although the size of each colony chosen was roughly the same, it is uncertain how much bacteria was transferred. This could have affected the results because the greater amount of bacteria present, the higher the chances for transformation and vice versa.

Lack of proper equipment was also a limitation. My school laboratory does not a refrigerator that can reach -20°C for proper storage of plasmid. This meant that the concentration of the plasmid decreased as time went on. Later transformations would be done with less plasmid per µl thus transformation efficiency would lower the overall average for transformation efficiency. Contamination may have been present in the samples, trials or stock. It is assumed that if any contaminants were present, the ampicillin present in the agar would kill it. However, there

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were cases where the contaminants were resistant to the ampicillin and this might cause the colony to be counted if it resembles the normal E.coli colony.

There is a chance that two CFU’s are placed together causing only one colony to form instead of two. This would lower the transformation efficiency count because fewer overall colonies are formed since the two CFU’s will merge into one colony. Therefore, there is an uncertainty if a single colony was made up from one CFU or more.

6.2 – Ways to Improve A wider range of temperatures should be studied to properly determine the optimum temperature for heat shocking. This is because there is a wide range between the temperatures of 37.0°C and 42.0°C and even between 42.0°C and 60.0°C. By experimenting with a greater range of temperatures, a more precise conclusion can be drawn. An electronic bacterial colony counter should be used to ensure greater accuracy at detecting the number of bacteria colonies. Manual detection (using the naked eye) is not suitable because smaller colonies are easily overlooked or colonies might be mistaken for air bubbles in the agar and vice versa. An electronic bacterial colony counter will improve the accuracy or at least help countercheck colony counts. The timing of this experiment can be improved. Due to unforeseen circumstances, this experiment took longer than planned which lead to plasmid degradation ,rendering more and more plasmids unusable. Hence, this experiment should be done immediately after purchase if proper refrigeration is not available. Some improvements are: all equipment on standby prior to the experiment and multiple water baths prepared for simultaneous heat shocks.

Replication of this experiment, either internally (more trials) or externally such as by another IB school, will allow for greater accuracy and reliability. This will lend credence and insight Page | 25

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particularly towards the temperature suggested for heat shock methodology. As more trials are conducted, the degree of error will be reduced with each subsequent experiment. If this experiment is done externally, environmental factors can be removed due to the change in environment.

6.3 – Further Investigations The potential of GFP should be explored next since the fundamentals of transformation have been established. Splicing the DNA sequence of GFP into a plasmid and then transferring the plasmid into bacteria is an exciting proposition. Glowing bacteria are easily identified; allowing bacterial infections to be studied in a live subject since staining is not required. GFP can also be expressed in eukaryotic cells such as human liver cells through the heat shock method. Termed as transfection, this will allow researchers to easily identify transfected cells by the glow. This is especially useful for understanding cancer and neurobiology. This study should further include other bacterial strains other than just E.coli. By testing out different bacteria, bacterial virulence and antibiotic resistance factors can be studied on a more detailed scale. This information would be helpful to healthcare professionals as steps can be taken to prevent the next outbreak of MRSA 16-like bacteria. Transformed bacteria can be “cured” to remove all plasmids contained within the bacteria. This is done mainly by disrupting plasmid replication or binding the plasmids using heterocyclic compounds rendering the plasmids unreadable. This can remove antibacterial resistance in bacteria leaving these bacteria vulnerable to antibiotics. Further research should be conducted on this topic to reduce the rate at which bacteria attain antibacterial resistance. The duration of the heat shock also plays a role in transformation efficiency. A longer heat shock but at a lower temperature might provide better results than a brief heat shock at a higher temperature. Reasons for this could be that at a longer heat shock period, the chance of 16

Multiple-resistant Staphylococcus au reus – a strain of Staph.a that is resistant to a wide range of antibiotics.

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exogenous plasmid entering the bacteria is prolonged. This could lead to alternative heat shock methods. Other factors which might increase transformation efficiency should also be studied. Different cations such as magnesium and potassium might have greater polar attraction to DNA molecules, thus aiding entry of plasmids into bacteria. Alternative transformation methods such as electroporation should be also be studied. This method is said to have higher transformation efficiency and an easier methodology although it requires highly specialized equipment and expensive materials compared to the heat shock method.

7.0 – Conclusion With enough supporting evidence, heat shock method is suggested to be done at 42.0°C because it is high enough to induce most bacteria to express of their heat-shock proteins. The more pore openings, the more plasmids are absorbed. Thus, the highest transformation efficiency is achieved.

At 37.0°C, transformation efficiency is lower compared to 42.0°C because the temperature at 37.0°C does not fully stimulate all HSPs contained within the bacteria. This results in a lower concentration of pore openings, which lowers the chances of plasmids entering the bacterial cell. At lower temperatures, the heat shock proteins are not fully stimulated. Transformation occurs at an efficiency too low for these temperatures to be suggested. At higher temperatures, the bacteria die due to protein denaturization. Again, transformation might occur since heat shock proteins are stimulated but transformation efficiency is low because most bacteria cannot survive.

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8.0 – Appendix

Appendix 1 – Genetically Modified Tobacco Plant

Figure A – Tobacco Plant glowing due to presence of firefly gene. Image taken from: <http://www.scionline.org/index.php/Genetically_Modified_Crops_in_Agriculture>

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Appendix 2 – Detailed Genetic Map for Plasmid pUC18

Figure B – Vector Sequence and Restriction Enzyme Map. Image taken from: <http://www.genscript.com/site2/document/5322_20080813063105.JPG>

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Appendix 3 – Preparation of Luria Broth agar Reagents: 1. 2.5 g – Yeast Extract 2. 5 g – Sodium Chloride 3. 5 g – Tryptone 4. 7.5 g – Bacteriological Agar 5. 500 ml – Water Methodology: All reagents above are measured using an electronic weighing machine and placed in a beaker. 500 ml of distilled water is measured and poured into the beaker. Heat the beaker using a Bunsen burner while stirring with a glass rod. Once all reagents are homogenized, placed them in a pressure bottle and sterilize using autoclave machine. While still liquid, pour the LB agar into petri dishes. Agar is left to cool.

Diagram C – Luria Broth on the left and Luria Broth Agar on the right

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Appendix 4 – ANOVA Test An ANOVA Test (ANalysis Of Variance Test) is used instead of the t-test because the t-test can only accommodate a total of 2 groups compared to the 5 groups (0°C, 25°C, 37°C, 42°C, 60°C) done in this experiment. • •

Null Hypothesis, H0 = Âľ1 = Âľ2 = Âľ3 = Âľ4 = Âľ5. This means that the change in temperature does not cause the Escherichia coli to have greater transformation efficiency. The Alternative hypothesis is defined as H1 = one or more means are different . This would allow us to infer that there is a significant difference between the groups, leading us to the conclusion that the heat shock method does affect the transformation rate.

All significances (Îą) for the following tests will be at 5% or 0.05. Assumptions for the ANOVA Test: 1. Observations are separate. 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. Homogeneity of variances; that is, the variances of all the groups are homogenous/equal. The ANOVA table is summarized in the table below: Source of Variation

Sum of Squares

Between Groups

SSb

Within Group

SSw

Total

SSt

df

(k – 1) (N – k)

Mean Squares (s2)

MSb2 =

đ?‘†đ?‘†đ?‘†đ?‘†đ?‘?đ?‘?

2 MSw =

đ?‘˜đ?‘˜ −1 đ?‘†đ?‘†đ?‘†đ?‘†đ?‘¤đ?‘¤

đ?‘ đ?‘ −1

F Ratio

MSb

MSw

Critical F

Fk – 1, N- k

P Value (computer generated)

(N – 1) Table D – 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

đ??Œđ??Œđ??Œđ??Œđ??›đ??›đ?&#x;?đ?&#x;?

Mean squares (variance) between groups

đ??Œđ??Œđ??Œđ??Œđ??°đ??°đ?&#x;?đ?&#x;?

Mean squares (variance) within group Table E – Legend for ANOVA Table

Temperature/ ¹ 0.5°C

0.0

25.0

37.0

42.0

60.0

x

x2

x

x2

x

x2

x

x2

x

x2

Result 1

–

–

–

–

–

–

2

4

–

–

Result 2

–

–

2

4

2

4

5

25

–

–

Result 3

–

–

–

–

2

4

2

4

–

–

Result 4

–

–

–

–

3

9

2

4

–

–

0

0

2

4

7

17

11

37

0

0

∑x

∑∑x2

Table F – Calculation for ANOVA (–) = No CFU’s found

Total summation, ∑x = 20 Summation for squared, ∑∑x 2 = 58

Page | 32

Jorrel Too Yu-Xann SSb = �

( 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑜𝑜𝑜𝑜0℃) 2 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

( 0) 2

( 2) 2

( 7) 2

CQV920(002206-017)

…+

( 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑜𝑜𝑜𝑜60℃) 2

( 11) 2

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 ( 0) 2

=� 4 + 4 + 4 + 4 + 4 �− = [0 + 1 + 12.25 + 30.25 + 0] − 20 = 43.5 − 20 = 23.5

SSw can be found by ∑∑x 2 − �

( 20) 2

� − 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇

( ∑x ) 2

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

20

( 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑜𝑜𝑜𝑜0℃ ) 2 𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

…+

( 𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑜𝑜𝑜𝑜60℃) 2

= 58 − 43.5 = 14.5

𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁 𝑜𝑜𝑜𝑜 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡

�

The total sum of squares ( SSt) is = SSw + SSb = 23.5 + 14.5 = 38.0 Calculating the mean squares/variance MSb = =

SS b

MSw =

𝑑𝑑𝑑𝑑 𝑏𝑏

23.5

=

4

= 5.875 The F ratio for this experiment is

=

=

=

𝑆𝑆𝑆𝑆𝑤𝑤

𝑑𝑑𝑑𝑑𝑤𝑤

14 .5 15

29 30

MS b

MS w

5.875 29 30

= 𝟔𝟔. 𝟎𝟎𝟎𝟎𝟎𝟎

F critical of F4,15 = 3.06 (refer to table below)

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Jorrel Too Yu-Xann

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Table G – Percentiles of F Distribution (5)

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Jorrel Too Yu-Xann

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Appendix 5 – Tukey’s Honestly Significant Difference (HSD) đ?‘€đ?‘€đ?‘€đ?‘€ HSD = đ?‘žđ?‘ž(đ?›źđ?›ź, đ?‘˜đ?‘˜, đ?‘ đ?‘ − đ?‘˜đ?‘˜)ďż˝ đ?‘¤đ?‘¤ đ?‘›đ?‘›

The values for Îą = 0.05 (represents the significant level) k = 5 (represents the total number of groups) N - k = 15 (total number of results - total number of groups) q = the value based on Îą, k, and N – k. = 4.37 Temperature , (Âą0.5°C)

0.0

Plate 1

–

–

–

–

–

2

4

–

Plate 2

–

2

4

2

4

5

25

–

Plate 3

–

–

–

2

4

2

4

–

Plate 4

–

–

–

3

9

2

4

–

0

2

–

7

17

11

37

–

Total

∑∑x2

25.0

37.0

42.0

60.0

Table H – Mean difference for tukeys HSD (–) = No CFU’s found

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Appendix 6 – Graphical Interpretation of Tukey’s HSD

Graph of Mean difference above the critical value 3

2.75

2.75

2.5

2.25 M e a n D i f f e r e n c e

2

1.5

1

0.5

0 0 – 25

0 – 37

0 – 42

0 – 60 25 – 37 25 – 42 25 – 60 37 – 42 37 – 60 42 – 60 Temperature Pair, °C Mean difference

Critical value

Graph I – Temperature pairs greater than the critical value. Page | 36

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Appendix 7 – Miscellaneous Diagrams

Figure J – Storage of extra agar in the lab refrigerator. All petri dishes are inverted to prevent any condensation from contaminating the agar. The temperature inside the fridge is 4.0°C.

Figure K & L – Pressure cooker used to autoclave suitable materials and equipment. The pressure cooker allows the water to boil above a 100°C which would be sufficient to kill all bacteria, thus promoting sterile condition and preventing contamination. The pressure cooker is left to run for about 5 minutes.

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Figure M – The freezer maintains a stable -4.0°C. Bio-degradation does occur because the necessary temperature for storage of plasmids is at -20.0°C. This error is dealt with under evaluations.

Figure N – During the experiment, all temperature sensitive materials are placed within an ice box filled with ice. This helps slow down biodegradation due to heat.

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Figure P– This is how the float is made. The microcentrifuge is placed in the middle of the Styrofoam float. It is ensured that the microcentrifuge is closed tightly and that the bacteria and plasmid solution is below the water level.

Figure Q – Transformed bacteria placed in an incubator. All petri dishes are inverted to prevent any moisture or condensation formed from dripping unto the agar. Notice that the incubator has a thermometer to ensure that the temperature is set at 37°C. It also has heat shields to prevent overheating and lowers light intensity.

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9.0 – Bibliography (1) McGraw-Hill. History of transformation. [book auth.] Leland Hartwell, Lee Hood, Michael Goldberg, Ann Reynolds, Lee M. Silver and Ruth C. Veres. Genetics: From Genes to Genomes-3rd Edition. New York: The McGraw-Hills Companies Inc., 2008. (2) Zimmer, Mark. History, Cool Uses and Nobel Prize. Green Fluorescent Protein [Online] [Cited: October 10, 2008] <http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm> (3) Stanley, Pamela. Transformation Methodology. Transformation of Bacteria by Heat Shock. [Online] November 24, 2005. [Cited: September 13, 2008] <http://stanxterm.aecom.yu.edu/wiki/index.php?page=Transformation_by_heat_shock> East Bay Biotechnology Education Program. Fluorescent Protein Powerpoint Presentation. [Online] [Cited: October 21, 2008] < http://www.ebbep.org/docs/flupro/fppowerpoint.pdf> Fort Hays State University Department of Chemistry. Recombinant DNA Technology. [Online] [Cited: October 21, 2008] <www.fhsu.edu/chemistry/twiese/360/slides/molbio.ppt> Zulkoski-Benson, Sarah. pGlo Bacterial Transformation. [Online] 2007. [Cited: October 21, 2008] < http://unlmbsc.wordpress.com/educator-and-student-materials-2/> A. Roe, Bruce. Protocol for Transformation and Transfection. Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma. [Online] [Cited: September 29, 2008] <http://www.genome.ou.edu/protocol_book/protocol_adxF.html> (4) Academic Press. Introduction of DNA into Bacteria. [book auth.] J.R. Saunders and V.R. Saunders. Methods in Microbiology Volume 29 – Genetic Methods for Diverse Prokaryotes. London: Academic Press, 1999 (5) McGraw-Hill. ANOVA. [book auth.] Jan W. Kuzma and Stephen E. Bohnenblust. Basic Statistics for the Health Sciences – 4th Edition. Singapore: Mayfield Publishing Company, 2001. Pope, Brian and M. Kent, Helen. High Efficiency 5 minute transformation of Escherichia coli. Nucleic Acids Research, Volume 24, No. 6, 1996. Oxford University Press, 1996. Page | 40

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Wikipedia, The Free Dictionary. Transformation (genetics). [Online][Cited: August 12, 2008] <http://en.wikipedia.org/w/index.php?title=Transformation_(genetics)&oldid=270219102>. iGEM 2007 Wiki. Boston University/Heat Shock Protocol. [Online][Cited: September 15, 2008] <http://parts.mit.edu/igem07/index.php/Boston_University/Heat_Shock_Protocol>.

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