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

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IB Biology EE on Bacteria Transformation by heat shock method using plasmid. Please cite and give proper reference to Seo Young if you use her work

IB Biology EE on Bacteria Transformation by heat shock method using plasmid. Please cite and give proper reference to Seo Young if you use her work

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  • 1. International Baccalaureate Diploma Program Extended Essay Biology Determining the effect of changing the atomic size of divalent cations ontransformation efficiency of the heat-shock protocol on Escherichia Coli DH5 strainCandidate Name: Seo Young MyaengCandidate Number: 002213-064Word Count: 3394
  • 2. Seo Young Myaeng Candidate Number: 002213-064Acknowledgements:I would like to acknowledge the following people for their support for making the investigation possible: Mr. Lawrence Kok Mr. Bob Johnson Jason Ji Ho Rhim Michael Seung-Hoon Shin Kelly Chaehyun Lee and Taejon Christian International School pg. 1/36
  • 3. Seo Young Myaeng Candidate Number: 002213-064 AbstractTransformation is a method used to change the genetic information of bacteria using acircular DNA called plasmid. Plasmid cannot normally enter the bacteria because both theplasmid and the bacteria cell membrane are negatively charged, and hence repel each other.However, by adding divalent cations to the bacteria, the cell membrane becomes neutralizedand permeable to the plasmid. Because of divalent cations’ essential role in transformation,the question of whether the type of cations used affects transformation efficiency was raised.Therefore, this investigation will focus on the research equation: To what extent doeschanging the atomic size of divalent cations affect transformation efficiency of the heat-shock protocol on Escherichia Coli DH5 strain?In the investigation, a heat-shock protocol on Eshcerichia Coli DH5 using pUC18 plasmidwill be performed. During the process, divalent cations of different sizes – Ca2+, Mg2+, andMn2+ – will be added in forms of chlorides. The pUC18 plasmid will make the bacteriaresistant to Ampicillin, a type of antibiotic. Thus, after transformation, the bacteria will beplated on LB-Ampicillin plate, an environment full of the antibiotic, to see whethertransformation was successful. The plates will be incubated for 48 hours at 37°C before theirtransformation efficiencies are calculated and compared.The result was that though cations of different sizes were used in different transformationprocesses, all transformations were successful. The values in different transformationefficiencies were not the same, but according to the Tukey’s HSD Test, not much significantdifference was found between results of certain cations, such as Ca2+ and Mn2+. From this, itcan be reflected that as long as divalent cations are used, transformation will be successful.In conclusion, despite the change in atomic size of cations, as long as the cations aredivalent, transformation will be successful.(294 words) pg. 2/36
  • 4. Seo Young Myaeng Candidate Number: 002213-064Table of Contents Abstract ................................................................................................................................. 2 1.0 Introduction .................................................................................................................... 4 1.1 Rationale of the Study ............................................................................................ 4 1.2 DH5 Bacteria ....................................................................................................... 5 1.3 pUC18 Plasmid ...................................................................................................... 6 1.4 History of Transformation ...................................................................................... 7 2.0 Hypothesis ....................................................................................................................... 8 3.0 Variables ......................................................................................................................... 9 3.1 Manipulated Variable ............................................................................................. 9 3.2 Responding Variable .............................................................................................. 9 3.3 Constant Variable ................................................................................................. 10 4.0 Materials and Methods ................................................................................................ 11 4.1 Overall Methodology ........................................................................................... 11 4.2 Preparation of LB and LB-Ampicillin Plates ....................................................... 12 4.3 Preparation of Salt Solutions ................................................................................ 12 4.4 Culturing Bacteria Stock ...................................................................................... 12 4.5 Transformation ..................................................................................................... 13 5.0 Data Collection ............................................................................................................. 14 5.1 Raw Data .............................................................................................................. 14 5.1-1 Qualitative Data ................................................................................... 14 5.1-2 Quantitative Data ................................................................................. 16 5.2 Calculation of Transformation Efficiency............................................................ 17 5.3 Data Analysis ....................................................................................................... 18 6.0 Statistical Analysis........................................................................................................ 20 6.1 ANOVA Test........................................................................................................ 20 6.2 Tukey’s HSD Test ................................................................................................ 21 7.0 Evaluation ..................................................................................................................... 22 7.1 Explanation........................................................................................................... 22 7.2 Uncertainties and Limitations .............................................................................. 23 7.3 Ways to Improve .................................................................................................. 25 7.4 Further Investigation ............................................................................................ 26 8.0 Conclusion ..................................................................................................................... 27 9.0 Appendix ....................................................................................................................... 28 10.0 References ................................................................................................................... 36 pg. 3/36
  • 5. Seo Young Myaeng Candidate Number: 002213-064 1.0 Introduction 1.1 Rationale of the StudyDuring transformation, bacteria cannot take in plasmid by itself due to repulsion. Therefore,the heat-shock method is used to make the bacteria more permeable to plasmid. First, divalentcations 1 are added in forms of chloride solutions. The standard chloride used duringtransformation is calcium chloride or CaCl2. The solution will then dissociate into Ca2+ andCl- ions. Ca2+ will then neutralize the negative charge on cell wall and membrane so that theplasmid will no longer be repelled, and thus enter the bacteria easily. Figure 1: Role of CaCl2 in transformation[1]However, just because Ca2+ is commonly used, it does not mean it is not the only type ofcations that may be used. There may be situations where there is a lack of CaCl2. Then it willbe necessary to know whether other divalent cations can be used to perform transformation.Through this investigation, I will use cations of the same charge (2+) but of different atomicsizes to observe whether it is possible to transform bacteria using solution other than CaCl2.Therefore my research question will be: To what extent does changing the atomic size ofdivalent cations affect transformation efficiency of the heat-shock protocol onEscherichia Coli DH5 strain?1 Molecules with the charge of 2+ pg. 4/36
  • 6. Seo Young Myaeng Candidate Number: 002213-064 1.2 DH5 Bacteria [2] Figure 2: Magnified image of DH5 [3 ]DH5 is a type of Escherichia Coli2 bacteria that will be used in the investigation. Thegeneral E. Coli bacteria are found in the lower intestines of endotherms3, and rarely causeharm other than through food poisoning. The bacteria can even be favorable to humans[4]since they produce vitamin K and are used in medicines for gastroenterological diseases4.Other than these general traits of E. Coli bacteria, the DH5 have their own specificcharacters that are important for this investigation. First, they have relatively thinpeptidoglycan layer5 and can allow plasmid to enter the cell more easily. This will helplessen the possibility of error from unsuccessful transformation. Also, DH5 can uptakelarge plasmids[5]. This is a crucial quality since the whole processes of transformation requiresthe bacteria to uptake plasmids and go through a genetic change.2 Which is also called E. Coli3 Warm-blooded animals4 A type disease on the digestive systems5 An outer layer, or barrier, of the bacteria that controls materials from entering pg. 5/36
  • 7. Seo Young Myaeng Candidate Number: 002213-064 1.3 pUC18 Plasmid Figure 3: Image of plasmid and bacterial DNA of a bacterium[5]A plasmid is a circular double-stranded DNA molecule that is usually used in genetics.Plasmid can be classified into two types: natural and artificial. The type of plasmid that willbe used in this investigation is pUC18, an artificial or genetically engineered plasmid. Figure 4: Image of pUC18 plasmid molecule[6]As shown in figure 4, pUC18 contains different types of genetic information, including onefor lacZ6. The lacZ and lac promoter7 allow pUC18 to recombine and transform the geneticinformation of a foreign DNA[7]. More importantly, pUC18 contains ampR 8 and thus isresistant to Ampicillin9. This character is essential to this investigation since the wholetransformation will be focused on making the bacteria resistant to Ampicillin. The existenceof ampR will allow the plasmid to be uninfluenced by the Ampicillin extant in LB-Ampicillinplate[8].6 A type of reporter gene, or gene that is easy to monitor7 Promoter of lacZ8 an antibiotic resistant gene9 Antibiotic used to treat bacterial infection pg. 6/36
  • 8. Seo Young Myaeng Candidate Number: 002213-064 1.4 History of TransformationTransformation[8] is a process that can alter the genetic information of an organism. It can becarried out by adding plasmid to the DNA that needs to be altered. Then, the two types ofDNA will recombine to form new genetic information.Frederick Griffith’s experiment[9] was the prototype of the transformation process. In 1928, hepresented that harmless Streptococcus pneumoniae bacteria could be turned lethal throughexposure to heat-killed virulent strains. Through this, Griffith hypothesized that a factor fromthe heat-killed virulent strains turned the Streptococcus pneumonia harmful. Figure 5: Diagram of Frederick Griffith’s experiments [9]After Griffith, Oswald Avery, Colin MacLeod, and Maclyn McCarty discovered that DNAwas responsible for making the bacteria strain transform[8]. They named the process of intakeof DNA by bacteria “transformation.” pg. 7/36
  • 9. Seo Young Myaeng Candidate Number: 002213-064 2.0 HypothesisThe purpose of this investigation is to determine the effect of the divalent cations’ size ontransformation efficiency. In order to change the cations’ size, cations of different atomicmass will be used. This is because atomic mass is determined by the sum of the number ofprotons and the number of neutrons10 that compose the atoms. Atomic Mass 2+ Mg 24.3 Ca2+ 40.1 Mn2+ 54.9 Table 1: the different divalent cations and their atomic massDuring transformation, a plasmid must enter the bacteria in order to change their geneticinformation. However, because both plasmid and the bacteria cell membrane are negativelycharged, the plasmid will be repelled and prevented from entering. Thus, transformation willless likely to be successful. However, when a chloride solution is mixed with bacteria, it willdissociate into cations and chloride ions. The cations will neutralize the negative charges onbacteria cell membrane. Therefore, the plasmid will no longer be repelled and can enter thebacteria to change the genetic information, hence leading to successful transformation. Cations neutralize the negative charges Plasma Membrane Figure 6: Diagram of how plasmid goes through plasma membrane when cations are usedIn this investigation, because all three kinds of cations have the charge of 2+, they will beable to neutralize the barriers to similar extents. This would mean in all transformationprocesses, the plasmid will be able to successfully enter the bacteria. Thus, it can be deducedthat despite the different sizes of the three cations, the transformations will be successful.10 Both protons and neutrons are (sub-)particles that make up an atom pg. 8/36
  • 10. Seo Young Myaeng Candidate Number: 002213-064 3.0 Variables 3.1 Manipulated VariableThe atomic size of divalent cations, which will be altered by using cations of differentatomic mass (Ca2+, Mg2+, Mn2+) In order to use cations of different atomic sizes, 0.1M chlorides of cations withdifferent atomic masses – CaCl2, MgCl2, and MnCl2 – will be used. 3.2 Responding VariableThe transformation efficiency of E. Coli strain After successful transformation, the bacteria will be able to survive on a LB-Ampicillin plate, an antibiotic environment. By using the following method, transformationefficiency for bacteria transformed with different cations will be obtained: 1111 Refer to Appendix 1 for further information pg. 9/36
  • 11. Seo Young Myaeng Candidate Number: 002213-064 3.3 Constant Variables- Type of bacteria used:In all transformation processes, E. Coli strain from the same bacteria stock will be used.- Type of plasmid used:For all trials of transformation, 5µl of pUC-18 plasmid will be used.- Type of Ampicillin used:The same antibiotic Ampicillin will be used to make the LB-Ampicillin plates.- Concentration of chlorides:The concentrations of all chlorides will be kept constant to 0.1M.- Period of Incubation:After transformation, all plates with the bacteria will be incubated for 48 hours.- Temperature of Incubation:The incubation temperature will be kept constant to 37°C, which is within the ideal range oftemperature for bacteria survival. pg. 10/36
  • 12. Seo Young Myaeng Candidate Number: 002213-064 4.0 Materials and Methods 4.1 Overall Methodology Plates (4.2) Preparing LB and LB Ampicillin Plates Salt Solutions (4.3) Preparing salt solutions Culturing Bacteria Stock (4.4) Culturing E. Coli bacteria stock Preparation of Chlorides Preparation of CaCl2, MgCl2, MnCl2 of the same concentration Transformation (4.5) Inserting bacteria into chlorides (250µl) Ice shock (5min) Inserting plasmid (5µl) Ice shock (15min) Heat shock (42 , 90sec) Ice shock (1min) Inserting Luria broth (250µl) Leaving at room temperature (15min) Streaking pg. 11/36
  • 13. Seo Young Myaeng Candidate Number: 002213-064 4.2 Preparation of LB and LB-Ampicillin Plates 250ml of LB agar is prepared12 The table below shows processes to prepare either LB or LB-Ampicillin plates LB Plates LB-Ampicillin Plates Agar is evenly poured into petri dishes  Agar is cooled until 57  4ml of Ampicillin is added to the agar  Agar is slowly swirled for Ampicillin to mix  Agar is poured evenly into petri dishes After agar solidifies, dishes are closed and turned upside down The dishes are stored in refrigerator at 4 4.3 Preparation of Salt Solutions Serial dilution is used to prepare 0.1M of CaCl2, MgCl2, and MnCl213 4.4 Culturing Bacteria Stock 200µl of LB broth is prepared14 Using a sterile loop, a colony of E. coli is scooped and added to the LB broth The loop is twirled to assure they are mixed well The LB broth is incubated at 37°C for 24 hours Using a new sterile loop, small amount of the LB broth is spread on a LB plate The plate is closed and sealed using parafilm, and is incubated at 37°C for 24 hours12 Refer to Appendix 2 for further information13 Refer to Appendix 3 for further information14 Refer to Appendix 4 for further information pg. 12/36
  • 14. Seo Young Myaeng Candidate Number: 002213-064 4.5 Transformation Label 6 microcentrifuge tubes “+plasmid” and three tubes “–plasmid” Using micropipette, 250µl of ice-cold CaCl2, MgCl2, and MnCl2, is each added to two “+plasmid” tubes and one “-plasmid” tube Using sterile loop, remove a colony of E. Coli from bacteria stock15 Add the colony to CaCl2 of a “+plasmid” tube Using vortex, assure the solution is mixed well Using centrifuge, suspend the solution down Steps 3 to 6 are repeated for the other 8 tubes All tubes are placed in ice for 5 minutes16 Using micropipette, in each “+plasmid” tube, 5µl of plasmid is added and mixed well All tubes are left in ice for 15 more minutes All tubes are removed from ice and are placed at 42 water bath for 90 seconds17; it is assured no water enters the tubes Immediately, all tubes are returned to ice for 1 minute18 All tubes are removed from ice Using micropipette, 250µl of Luria broth is added to every tube The tubes are left at room temperature for 15 minutes Using micropipette, 100µl of each tube’s solution is placed on a LB-Ampicillin plate Using micropipette, 100µl of each “-plasmid” tube’s solution is placed on a LB plate Using sterile loop, the solutions on the plates are streaked19 All plates are closed, turned upside down, and incubated at 37 for 48 hours15 Bacteria stock from step “4.4 – Culturing Bacteria Stock”16 Process of ice-shock17 Process of heat-shock; used to open pores of bacteria membrane so that plasmid can enter the cell18 Process of ice-shock; used to allow membrane pores to close and prevent plasmid from escaping19 Refer to Appendix 5 for further information pg. 13/36
  • 15. Seo Young Myaeng Candidate Number: 002213-064 5.0 Data Collection 5.1 Raw Data 5.1-1 Qualitative DataNote: The following images are bacteria plates after 48th hour of incubationMgCl2 MgCl2 Plate 1 MgCl2 Plate 2 Plate Number Plate 1 Plate 2 Number of Colonies (CFU) 28 4 Observations It can be easily noticed that the plates have small numbers of bacteria colonies. The colonies are also small and cannot be seen unless observed closely and carefully.CaCl2 CaCl2 Plate 1 CaCl2 Plate 2 Plate Number Plate 1 Plate 2 Number of Colonies (CFU) 150 172 Observations The number of colonies formed on the two plates is great. Most colonies are thick and big enough to be seen easily. pg. 14/36
  • 16. Seo Young Myaeng Candidate Number: 002213-064MnCl2 MnCl2 Plate 1 MnCl2 Plate 2 Plate Number Plate 1 Plate 2 Number of Colonies (CFU) 80 152 Observations Many colonies have formed on the plates. Most colonies are thick and big enough to be seen easily.Negative Control LB-Ampicillin Plates LB Plates Type of Plate LB-Ampicillin LB Type of MgCl2 CaCl2 MnCl2 MgCl2 CaCl2 MnCl2 Chloride Number of - - - N/A N/A N/A Colonies Observations No colonies formed at all Though it is difficult to observe from the on the plates image, a countless number of bacteria colonies were formed. The plates are full of streaks of bacteria colonies pg. 15/36
  • 17. Seo Young Myaeng Candidate Number: 002213-064 5.1-2 Quantitative Data Number of Bacteria Colony (CFU20) Plate 1 Plate 2 Average MgCl2 28 4 16 CaCl2 150 172 161 MnCl2 80 152 116 Negative Control - - - (LBA plate) Negative Control N/A N/A N/A (LB plate) Table 2: Number of bacteria colonies on each plate on the 48th hourNote:“-“: no colonies formed“N/A”: the colonies were uncountable20 Colony-forming unit: unit for measurement of bacterial number pg. 16/36
  • 18. Seo Young Myaeng Candidate Number: 002213-064 5.2 Calculation of Transformation EfficiencyThe equation for transformation efficiency is:For successful calculation, the total mass of plasmid used needs to be found out as below:Where: µlThe value of “total mass of plasmid used” is unknown, but can be found as shown below:Thus: µgTherefore: Chloride Average number of colonies Transformation Efficiency (CFU/µg) (CFU) MgCl2 16 CaCl2 161 MnCl2 116 Table 3: Calculation of transformation efficiency for different chlorides pg. 17/36
  • 19. Seo Young Myaeng Candidate Number: 002213-064 5.3 Data Analysis Transformation Efficiency/colonies µg-1 against Type of Cations 350 300 Trnasformation Efficiency/CFU µg-1 250 200 Magnesium Calcium 150 Manganese 100 50 0 Magnesium Calcium Manganese Type of Cations Figure 7: Diagram of transformation efficiency against type of divalent cations pg. 18/36
  • 20. Seo Young Myaeng Candidate Number: 002213-064 Transformation Efficiency/colonies µg-1 against Atomic Mass 350 y = -0.7985x2 + 69.781x - 1192.1 300 R² = 1 Transformation Efficiency/CFU µg-1 250 200 150 100 50 0 0 10 20 30 40 50 60 Atomic Mass of Cations Figure 8: Diagram of transformation efficiency against atomic mass pg. 19/36
  • 21. Seo Young Myaeng Candidate Number: 002213-064 6.0 Statistical Analysis 6.1 ANOVA TestANOVA (Analysis of Variance) Test21 is one of the ways to compare the resulting data fromdifferent cations and observe whether there is a significant difference in the results or not. Forthe calculations, raw data on the number of colonies was used. Null Hypothesis (H0): there is no significant difference among different chlorides Alternate Hypothesis (HA): there is significant difference among different chloridesIf F ratio > F critical = Null Hypothesis is rejected and Alternate Hypothesis is made valid.Note: Calculations were done using the Excel Program.Source of Variation Sum of df Mean squares F Ratio Critical F P Value Squares (s2) Between Groups 22,033 2 11,016 10.59 9.55 0.0440 Within Groups 3,122 3 1,040 Total 25,155 5 Table 4: Results of the ANOVA TestBecause F ratio (10.59) > F critical (9.55), the Null Hypothesis is rejected and the AlternateHypothesis is accepted.Therefore, there is significant difference between groups.21 Refer to Appendix 6 for further information and calculation pg. 20/36
  • 22. Seo Young Myaeng Candidate Number: 002213-064 6.2 Tukey’s HSD TestThe Tukey’s HSD (Honestly Significant Difference) Test22 can also be used to analyzeresults of this investigation. This test will compare all possible pairs of groups to see whichgroup pair is greater than the critical value. Tukey’s HSD Test Critical Value: Where Cations Atomic Mass Mean Difference Critical Value (of Average CFU) 135 135 135 Table 5: calculation and presentation of mean difference and critical valueIn the ANOVA Test, results show that there is significant difference between groups.However, this was when putting all three results together. The Tukey’s HSD Test directlycompares two cations at a time.According to the test, the following pair has a mean difference greater than critical value.Simultaneously, the fact that the pair of are close to the criticalvalue could also be considered (Then both pairs include the cations, magnesium.).22 Refer to Appendix 7 for further information and calculation pg. 21/36
  • 23. Seo Young Myaeng Candidate Number: 002213-064 7.0 Evaluation 7.1 ExplanationThe results of this investigation were that transformation efficiency from transformationusing calcium cations was the greatest, manganese cations the second greatest, andmagnesium cations the lowest. Though the values of transformation efficiency are not equalfor the different sized cations, all transformations for different cations were successful. Thus,it can be claimed that despite atomic size of cations, as long as they are divalent,transformation will successfully occur. This can be explained by the fact that all three cationshave the same charge of 2+ and the same role of neutralizing bacteria membrane for plasmidto enter the bacteria. Because of these common qualities, all cations are able to neutralize themembrane to a similar extent and thus allow successful transformation to occur.According to the ANOVA Test, the transformation efficiencies among different cations havesignificant differences. However, the ANOVA Test compares the results from all threegroups simultaneously while the Tukey’s HSD Test compares two groups at a time. TheTukey’s HSD Test states that there is no significant difference between calcium andmagnesium’s transformation efficiencies. Also, the mean difference between magnesium andmanganese is smaller than but close to the critical value. Thus, the possibility of experimentalerrors behind the results of manganese or magnesium cations may be considered. Thesestatistics help show that the transformation efficiencies for divalent cations of differentatomic sizes are not of great difference and that atomic size may not be significant cause ofdifferent transformation efficiencies.Therefore, the results of this investigation can support the explanation and claim thatregardless of the sizes of the cations, as long as it is divalent, successful transformation willoccur. pg. 22/36
  • 24. Seo Young Myaeng Candidate Number: 002213-064 7.2 Uncertainties and Limitations Amount of stock bacteria added: CaCl2, MgCl2, and MnCl2 were each mixed with a colony of E. Coli scooped from the stock bacteria. Though it was attempted to keep the amount of bacteria constant, the specific amount remains different. This difference in amount of bacteria may cause significant difference in amount of transformed bacteria and also in the amount of bacteria spread on LB-Ampicillin plate. Amount of bacteria plated: It cannot be certain that the amount of bacteria plated after transformation were equal. Since bacteria have the tendency to clump together, the 100µl of plated solution may not have contained equal amount of bacteria. If so, then the number of bacteria on the LB-Ampicillin plates would have differed and hence influenced the overall result of the investigation. Contamination of bacteria during transformation: During the transformation process, the bacteria solution was exposed to open air and could possibly have been contaminated. This means that other bacteria from open air may have been transformed together. When counting the number of colonies transformed, the contaminant may also have been counted together and affect the result of the investigation. Also, the bacteria solution may have been contaminated because of long exposure to open air while being plated on LB- Ampicillin plate. If the contaminant is resistant to Ampicillin, then it would have been incubated and counted together with E. Coli bacteria, thus affecting the overall result. Condition of the chlorides: The MgCl2 and MnCl2 used during the investigation were man-made. Therefore, there is a possibility that an error occurred when creating the chlorides. More error could have occurred if the magnesium or manganese did not dissolve in the chlorides well and hence are not mixed well within the solutions. Then, unequal amounts of cations would have been used during the investigation, and hence influence the overall results. pg. 23/36
  • 25. Seo Young Myaeng Candidate Number: 002213-064 Condition of LB-Ampicillin Plates: The conditions of different plates used to spread transformed bacteria could influence the results, too. The LB-Ampicillin plates used were manmade and there is the possibility of the LB-broth not being mixed thoroughly. If so, each plate would contain different amount of LB broth, or bacteria food. Then the bacteria would have been incubated in “different environments” and influence the results. Lack of trials and variables: In this investigation, there were only three manipulated variables and for each variable only two LB-Ampicillin plates were created. The increase in number of manipulated variables would have allowed better support and understanding of results. It will also show whether the change in atomic size truly has a specific pattern of relationship with transformation efficiency. Moreover, with greater number of trials for each variable, there would have been more results to decrease the experimental error and allow better comparison of the results. pg. 24/36
  • 26. Seo Young Myaeng Candidate Number: 002213-064 7.3 Ways to Improve Making of chlorides: In this investigation, CaCl2 was a pre-made solution while the other two were handmade. The condition of CaCl2 could not be controlled but is most likely to have been stable. However, because both MgCl2 and MnCl2 were handmade solutions, it is possible for the cations to not have been mixed well with the chloride. Hence, the solutions would not have been as stable as CaCl2. The investigation may be improved by assuring the chemicals are mixed well and are made into homogenous solutions. Increase in number of trials: In this investigation, because only two trials were done for each cation, it is unsure whether the results are experimental errors or are actual valid values. With greater number of trials, it will be possible to compare the results from the different trials and evaluate whether the results are valid and reliable. Increase in number of variables: In this investigation, there were only three manipulated variables. Though a conclusion can be drawn with these results, if more manipulated variables were used, then the results could turn out different. The increase in number of variables would have allowed clearer views and understandings of the results, and added to the validity and reliability of the investigation. Adding similar amounts of bacteria: During the transformation process, several errors occurred from lack of caution when adding bacteria. With more caution and care, these errors can be improved and thus obtain more reliable results. For example, when adding stock bacteria to the chlorides, with thorough measurement of the size of the colony being added, error from the difference in amount of bacteria added can be decreased. Also, by the end of the experiment, LB broth mixed with transformed bacteria has to be added on LB-Ampicillin plate using micropipette. In this process, by simply mixing the solution again before plating, it can be assured that the bacteria is evenly mixed within the solution and thus be able to plate similar amount of bacteria on each plate. pg. 25/36
  • 27. Seo Young Myaeng Candidate Number: 002213-064 7.4 Further InvestigationGene transformation is not a technique that can only be applied to bacteria. It is an efficientmethod to change the genetic information of other organisms for the benefits of humans. Forexample, currently, maize crops’ genetic information is being changed to increase crop yield.Gene transformation can also be used on other organisms to gain information on geneticdiseases or ways to improve certain diseases. Because the technique can be used for variousreasons, it is crucial for efficient gene transformation to occur. In this investigation, threedifferent variables were used to determine whether different atomic size of divalent cationsaffect transformation efficiency. The result was that despite the atomic sizes, as long as thecations are divalent, transformation will occur. Through further investigations on thisexperiment, other variables necessary for successful transformation may be found.One further investigation that could be carried is determining whether the same rule appliesfor bacteria other than E. Coli. This investigation was a success due to the usage of E. Coli,but it does not mean that the hypothesis is true for all types of bacteria or organisms. It ispossible that different results will come out for different bacteria. It is possible to carry outthe same investigation as this one but alter the E. Coli into different organism. Thus thedifferent effects of transformation on different bacteria and organisms may be found out.Another possible further investigation that could be carried out would also be to determinethe relationship between change in cations’ size and transformation efficiency. The onlydifference would be that the cations will have a different charge. During this specificinvestigation, all cations were divalent or had the charge of 2+. However, it is unknownwhether similar observations can be made for cations of different charges, such as 1+ and 3+.Thus, it will be possible to determine whether the same observation made in this investigationwill be applicable for cations of different charges. Through this further investigation, theeffects of cations’ charge on transformation efficiency may also be determined. This wouldbe done by comparing the results from the further investigation and from this specificinvestigation. pg. 26/36
  • 28. Seo Young Myaeng Candidate Number: 002213-064 8.0 ConclusionDuring bacteria gene transformation, a chloride with divalent cations is added to make thebacteria’s cell membrane more permeable to the plasmid. The plasmid, which enters thebacteria cell will then be able to alter the genetic information of bacteria and hence make itgo through transformation. Normally, calcium chloride is used during transformation. But inthis investigation, chlorides of divalent cations with different atomic mass were used toinvestigate the relationship between the atomic size and transformation efficiency.Through the experiments, it was observed that though the values of transformation efficiencywere different for different divalent cations, transformation still occurred. The explanationbehind this is that because all three cations had equal charge of 2+, they were able toneutralize the bacteria membrane to a similar extent to allow plasmid to enter the cell. Thus,it can be claimed that despite the atomic size of cations, as long as it is divalent,transformation will be successful. pg. 27/36
  • 29. Seo Young Myaeng Candidate Number: 002213-064 9.0 AppendixAppendix 1) Calculation of “amount of plasmid plated in ”Appendix 2) Preparation of LB agar  Using an electronic balance, 2.5g of tryptone, 1.25g of yeast, 2.5g of NaCl, and 3.75g of agar are measured and prepared  The reagents are added to a 250ml conical flask along with 250ml of distilled water  The flask’s mouth is covered with a small beaker  The flask is placed inside an autoclave and is autoclaved until 17 psi  The autoclave is turned off and is left until the pressure is released  The conical flask is removed from the autoclave and is slowly swirled in order to prevent the agar from solidifyingAppendix 3) Process of 10-fold serial dilution 10cm3 distilled water 100ml of 1M 0.1M Cacl2, MgCl2, or MnCl2 Figure A) Diagram of 10-fold serial dilutionAppendix 4) Preparation of LB broth  10g of Luria-Bertani powder are added to 400ml of distilled water  The solution is mixed well and is autoclaved pg. 28/36
  • 30. Seo Young Myaeng Candidate Number: 002213-064Appendix 5) Method of StreakingSolutions and/or bacteria are streaked on to plates as following[10]: Figure B) Diagram on method of streaking bacteria pg. 29/36
  • 31. Seo Young Myaeng Candidate Number: 002213-064Appendix 6) ANOVA Test [11]The Analysis of Variance (ANOVA) Test allows a comparison of 3 or more groups, such asthe ones used in this experiment (Mg2+, Ca2+, and Mn2+). By using the ANOVA Test, eitherof the two following hypothesis will be accepted: 1. The Null Hypothesis,This is when the change in cations’ atomic size, or atomic mass, does not cause change intransformation efficiency of E. Coli. 2. The Alternate Hypothesis, one or more means are differentThis is when it can be deduced that there is significant different between the groups’ resultsand thus the change in atomic mass does bring change in transformation efficiency.The ANOVA Test is also taken place with the following assumptions: 1. Results from one group are independent from the other; they do not affect the other groups’ results. 2. Observations from each group follow the normal distribution statistics. 3. Variances of all groups are equal pg. 30/36
  • 32. Seo Young Myaeng Candidate Number: 002213-064Below is an example of an ANOVA Test table and its summary:Source of Sum of df Mean squares F Critical F P ValueVariation Squares (SS) (s2) Ratio Between SSb (computer Groups generated) Within SSw Groups Total SSt Table A) Table on summary of ANOVA Test Notation Meaning SSb Sum of squares between groups SSw Sum of squares within groups SSt Sum of squares in total df Degree of freedom k Total number of groups (= 3) N Total number of results (= 6) Mean squares / Variance between groups Mean squares / Variance within groups Table B) Legend for Table A Mg Ca Mn x x2 x x2 x x2 Plate 1 28 784 150 22500 80 6400 Plate 2 4 16 172 29584 152 23104 32 322 232 800 52084 29504 Total Sum of 586 Total Sum of 82388 Table C) Calculation Step for ANOVA Test pg. 31/36
  • 33. Seo Young Myaeng Candidate Number: 002213-064Calculation of :Calculation of :Calculation of :Calculation ofCalculation of :Calculation of F ratio:F Critical Value ofNote: value was achieved from table below where and = . pg. 32/36
  • 34. Seo Young Myaeng Candidate Number: 002213-064 Table B) the F value or F distribution of ANOVA test [12] pg. 33/36
  • 35. Seo Young Myaeng Candidate Number: 002213-064Appendix 7) Tukey’s HSD Test[13] Tukey’s HSD Test Critical Value: WhereThe HSD critical value will be compared with the differences of colony numbers betweentwo cations results. If the differences are larger than the critical value, then it can be claimedthat the results’ differences are significant. Table C) Table of q values[14]Note: df error represents the value pg. 34/36
  • 36. Seo Young Myaeng Candidate Number: 002213-064Graphical Interpretation of Tukey’s HSD Mean Difference Above Critical Value 200 150 Mean Difference 100 Mean Difference 50 Critical Value 0 Mg-Ca Mg-Mn Ca-Mn Cations Pair Figure C) Graph of cations pair greater than critical value pg. 35/36
  • 37. Seo Young Myaeng Candidate Number: 002213-064 10.0 References[1] http://biochemistry.yonsei.ac.kr/biochem_molecular/gene_cloning_17.php[2] http://www.pnas.org/content/101/42/15027/F2.expansion.html[3] “E. Coli Genotypes.” Wikipedia. 2011. Web. <http://openwetware.org/wiki/E._coli_genotypes#DH5.CE.B1>.[4] “Escherichi Coli.” Wikipedia. 2011. Web. <http://en.wikipedia.org/wiki/Escherichia_coli>.[5] http://en.wikipedia.org/wiki/File:Plasmid_(english).svg[6] http://www.mun.ca/biology/scarr/Plasmid_pUC18.html[7] “Plasmid pUC18.” Memorial University. 2010. Web. <http://www.mun.ca/biology/scarr/Plasmid_pUC18.html>.[8] “Transformation (genetics).” Wikipedia. 2011. Web. < http://en.wikipedia.org/wiki/Transformation_(genetics)>. https://filebox.vt.edu/users/mahogan2/Filebox%20Portfolio/Webquest%20for%20DNA_fil[9]es/image002.jpg http://www.marine.csiro.au/microalgae/methods/support%20or%20original%20files/micro[10]a3.jpg[11] Yu-Xann, Jorrel Too. Determining the optimum temperature that produces the highest transformation efficiency rate using the heat shock transformation method on modified Escherichia coli strain. 2008. Print. http://www.velocebit.com/Courseware/Statistics/Common%20Files/Analysis%20of%20Va[12]riance/Text%20Figures/Table%20A6001.jpg[13] Yu-Xann, Jorrel Too. Determining the optimum temperature that produces the highest transformation efficiency rate using the heat shock transformation method on modified Escherichia coli strain. 2008. Print.[14] http://www.tcnj.edu/~ruscio/Guide%20to%20SPSS%20for%20Windows%20S11.pdf pg. 36/36