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 DH5Îą strain.
Candidate Name: Ji Ho Rhim Candidate Number: 2213-081 Word Count: 3667
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 2+
in the Ca 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 4
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 competency 1 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 DH5ι strain.
1 2 3
Being able to uptake new genetic information See Appendix B Will be further discussed later on in the investigation 5
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 6
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 AveryMacLeod-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 6 7
Harmless Harmful Virulent strain of bacteria 7
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.
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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 9
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 11
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 DH5Îą 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
12
will 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 13
4.0
Methodology 4.1
Preparation of Ampicillin-LB agar plates 1. 1 L of LB agar
13
is 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 14
1.
200 Îźl of LB Broth
is 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 14
4.3
Heat Shock Transformation Procedure
Using a 50μl micropipette place 15μl of Bracteria-LB broth solution into each of the five microcentrifuges
Take the ice-cold CaCl of different concentrations and put 150μl in each of the different concentration into 5 different autocalved microcentrifuges
Label microcentrifuge tubes
Place in ice bath for 15 minutes
Add 5μl of the plasmid into the microcentrifuge
Vortex the micro centrifuge tubes
Incubate in ice for 15 minutes
Heat shock in 42 C water bath for 90 seconds
Incubate in ice for 10 minutes
Add 250μl of LB Broth into each of the centrifuges and vortex each of the centrifuges for one minutes
Take 100μl of each concentration three times and swab it accordingly with sterile cotton swabs to the three individually labeled LB ampicillin plate
Place the plates upside-down inside an incubator at 37 C and incubate for a day
Remove Petri dishes from incubator and count CFUs on a black background
Duplicate experiment
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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 6.) E. Coli Stock Plate – To check if bacteria is viable
Figure 7.) Positive Control Plate – To check if bacteria survive transformation protocol
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Figure 8.) Trial 1 CFU count of 0.02M (left) and 0.1M (right) CaCl2 washed E. Coli DH5Îą [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.
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Figure 9.) Trial 1 CFU count of 0.04M (left) and 0.08M (right) CaCl2 washed E. Coli DH5Îą [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.
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Figure 10.) Trial 1 (left) and Trial 2 (right) CFU count of 0.06M CaCl 2 washed E. Coli DH5Îą [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
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Figure 11.) Trial 2 CFU count of 0.02M and 0.10M CaCl2 washed E. Coli DH5Îą [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
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Figure 12.) Trial 2 CFU count of 0.04M (left) and 0.08M (right) washed CaCl2 washed E. Coli DH5Îą [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.
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5.1.2 Quantitative Data 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
Table 3.) Table of Countable CFUs in each plate
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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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Concentration of CaCl2 Solution, mol dm-3
Trial 1
Trial 2
Total Average CFUs formed
Transformation Efficiency, (colonies / Îźg)
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
Table 4.) Transformation Efficiencies for Trials 1 and 2 at different Concentrations of Calcium Chloride Solution
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Calcium Chloride Concentration/ mol dm-3 against Transformation Efficency/ colonies Îźg-1
Transformation Efficiency/ colonies Îźg-1
12
10
8
6
4
2
0 0.02
0.04
0.06
0.08
0.1
Calcium Chloride Concentration/ mol dm-3
Graph 1.) Calcium Chloride Concentrations against Transformation Efficiency 26
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.
ANOVA Source of Variation
Sum of Squares
df
Mean Squares
Between Groups
60.8
2.0
30.4
Within Groups
20.7
8.0
2.6
Total
25.2
12.0
F Ratio 11.7
P-Value Comp. Generated
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
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F Critical 4.5
Calcium Chloride Concentration, mol dm-3
Mean Difference
Critical Value
0.02-0.10 0.02-0.08 0.02-0.06 0.02-0.04 0.04-0.10 0.04-0.08 0.04-0.06 0.06-0.10 0.06-0.08
4 – 3.5 = 0.5 5.5 - 3.5 = 2 5.0 - 3.5 = 1.5 3.5 - 3.5 = 0 4-3=1 5.5 - 3.5 = 2 5 - 3.5 = 1.5 5-4=1 5.5 - 5 = 0.5
1.68 1.68 1.68 1.68 1.68 1.68 1.68 1.68 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. 29
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. 30
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 31
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 33
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 heatshock 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 â&#x20AC;&#x201C;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 â&#x20AC;&#x201C; pUC18
Diagram 3.) pUC18 Vector <Image Taken from http://www.genscript.com/vector/SD1162-pUC18_plasmid_DNA.html>
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