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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 using heat shock method

IB Biology EE on bacteria transformation using heat shock method

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  • 1. INTERNATIONAL BACCALAUREATE DIPLOMA PROGRAM EXTENDED ESSAY BIOLOGYDetermining the optimum temperature that produces the highest transformation efficiency rate using the heat shocktransformation method on modified Escherichia coli DH5 ૒ strain. by CHAEHYUN LEE Candidate Number: 002213-048 Word Count: 3, 636
  • 2. Chaehyun Lee 002213-048 Acknowledgement My extended essay couldn’t have finished without the support from: Mr. Lawrence, for guiding us and supporting us throughout the process. Jason Rhim and Michael Shin for sacrificing their time to helping us. Seo young Myaeng for supporting and encouraging me as my extended essay partner. Page 2 / 36
  • 3. Chaehyun Lee 002213-048 AbstractTransformation is the transferring of extracellular DNA into an organism. Most bacteria aren’t able totransform naturally because they are unable to absorb plasmids naturally. Thus, in order to allowbacteria to uptake DNA, heat shock method is necessary to artificially induce competence using asudden increase in temperature for a brief period of time. This research therefore attempts todetermine which temperature will allow for optimum transformation efficiency.Four different temperatures were tested: 9°C, 30°C, 42°C, and 51°C. The bacterium of choice was themodified Escherichia coli DH5 ∂ and the plasmid was pGREEN. Plasmid pGREEN allows bacteria to glowin yellow-green color and to become ampicillin-resistant. These transformed ampicillin-resistantbacteria are able to survive on LB-ampicillin (LBA) plates because they have resistance against ampicillin,which kills bacteria. However, untransformed bacteria are unable to survive on LBA plates. Therefore,any bacteria that were present on an LBA plate were said to be transformed. The transformationefficiency was calculated based on the number of colonies present after incubation overnight.At 9℃ and 51℃, transformation efficiency for the two temperatures was zero. At 30℃, thetransformation efficiency was calculated to be 150. At 42℃, transformation efficiency was 500. 42℃ hadthe highest transformation efficiency. Further statistical analysis using ANOVA test and Turkey’s HSDtest reveals that the temperature of 42°C is more significant (p< 0.05) and hence more efficientcompared to 30°C.Based on the evidence obtained during this investigation, it can be concluded that the optimumtemperature for heat shock method is 42℃. This temperature is high enough to activate most heatshock proteins and low enough to prevent proteins from denaturing.(274 words) Page 3 / 36
  • 4. Page 4 / 3663 ..... ...................................................................................................... ................................ YHPARGOILBIB 0.0192 ............ ...................................................................................................... ..................................... XIDNEPPA 0.982 ....... ...................................................................................................... ................................... NOISULCNOC 0.862 .......................................................................................................................................... snoitagitsevnI rehtruF 4.762 .................................................................................................................................................. evorpmI ot syaW 3.752 ............................................................................................................................ snoitatimiL dna seitniat recnU 2.742 ......................................................................................................................................... stluseR fo noitanalpxE 1.742 ............................................................ ................................................... ................................... NOITAULAVE 0.732 ................................................................................................................................................. tseT DSH s’yekruT 2.622 ........................................................................................................................................................... tseT AVONA 1.622 ..................................................................................................... ......................... SISYLANA LACITSITATS 0.602 ............................................................................................. ycneiciffE noitamrofsnarT fo noitaluclaC 1.2.502 ................................................................................................................................................... gnissecorP ataD 2.591 ..................................................................................................................................... ataD evitatitnauQ 2.1.581 ........................................................................................................................................ ataD evitatilauQ 1.1.581 ............................................................................................................................................. noitcelloC ataD waR 1.581 ..................................................... ................................................... .............................. NOITCELLOC ATAD 0.551 ...................................................................................................................................................... noitamrofsnarT 4.451 ................................................................................................................... airetcaB tnetepmoC fo noitaraperP 3.451 ........................................................................................................................... setalp pmA :BL fo noitaraperP 2.441 .................................................................................................................... ygolodohteM fo weiv revO lareneG 1.441 ... ...................................................................................................... ................................... YGOLODOHTEM 0.431 ............................................................................................................................................... elbairaV dellortnoC 3.321 ............................................................................................................................................ elbairaV gnidnopseR 2.311 ............................................................................................................................................ elbairaV detalupinaM 1.311 ............ ...................................................................................................... .................................... SELBAIRAV 0.301 ............................................................ ................................................... ................................... SISEHTOPYH 0.29 ............................................................................................................................................................ noitamrofsnarT fo sesU 4.2.18 ...................................................................................................................................................... noitamrofsnarT fo y rotsiH 3.2.17 ........................................................................................................................................ NEERGP dimsalP 2.2.16 .............................................................................................................................................. 5HD iloC .E 1.2.1 ૒6 ........................................................................................................................................................ noitamrofsnarT 2.15 .................................................................................................................................................. ydutS rof elanoitaR 1.15 ......................................................... ................................................... ................................. NOITCUDORTNI 0.14 .................................................................. ................................................... ......................................... STNETNOC3 ....................................................................................................................... ......................................... TCARTSBA stnetnoC fo elbaT002213-048 Chaehyun Lee
  • 5. Chaehyun Lee 002213-0481.0 Introduction1.1 Rationale for StudyThe transfer of new DNA into organisms has led to many improvements in the fields of science,especially medicine. Thus, I found transformation to be a worthy topic of research. The technique oftransformation in plants and animals are extremely complex and costly. However, gene transfer in E.Coli bacteria are relatively simple and suitable for school laboratories. Hence, E. Coli was chosen to gothrough heat shock transformation procedure.Although heat shock method is less costly than electroporation method1, which is another method toinduce competency 2of bacteria cells, heat shock3 protocol is not inexpensive as well. Thus, it is crucial toincrease the efficiency of transformation as much as possible.The heat shock method has several variations. I decided to investigate the impact of temperature ontransformation rate because the temperature directly affects the efficiency of heat shock, which is acrucial part of transformation protocol. The temperatures of 37℃ and 42℃ are known to be the mostcommon and temperatures found in scientific sources. It is important to find out which temperatureworks the best for transformation.Thus, by determining the optimum temperature for the heat shock methodology, I can help contributeto bacterial transformation research and to fellow students who have limited number of trials andlimited time.Therefore, my precise research topic is:Determining the optimum temperature that produces the highesttransformation efficiency rate using the heat shock transformation method onmodified Escherichia coli strain.1 A method that uses electrical shock to create temporary competency within the cell.2 The state of being able to uptake new genetic information3 A method that uses heat shock to temporarily allow cell membrane to be permeable Page 5 / 36
  • 6. Chaehyun Lee 002213-0481.2 Transformation 1.2.1 E. Coli DH5 ૒E. Coli DH5 ∂ is a harmless, rod-shaped bacterium [1]. One significant characteristic of E. Coli DH5 ∂ isthat it is a gram negative bacterial strain. Its gram negative structure is important in the process oftransformation because it allows transformation process to work. Figure 1: Difference between the structure of gram positive and gram negative bacteriaAs opposed to gram-positive bacteria, gram-negative bacteria have a thinner layer of peptidoglycan anda periplasmic space between the cell wall and the membrane [2]. On top of that, gram-negative bacteriahave no nuclear membrane.These characteristics allow gram-negative bacteria, E. Coli DH5 ∂ , to be highly transformable. Moreover,since E. Coli DH5 ∂ is a relatively harmless bacterium, it can be handled safely during experiments. Page 6 / 36
  • 7. Chaehyun Lee 002213-0481.2.2 Plasmid PGREENPlasmid is a vector and a self-replicating circular strand of DNA that can be modified. Once the vector isabsorbed by the organism, certain proteins that are not normally synthesized will be present. Theseproteins may confer antibiotic resistance.The selectable marker of plasmid pGREEN is beta-lactamase. pGREEN’s color marker is mutant GreenFluorescent Protein4 fusion gene. This allows transformed bacteria to glow in yellow-green color. Thephenotypes of pGREEN’s transformants are ampicillin-resistant and yellow-green colonies. Figure 2: Plasmid map of pGREEN4 Green Fluorescent Protein (GFP) is a protein that exhibits bright greenfluorescence when exposedto ultraviolet blue light. Page 7 / 36
  • 8. Chaehyun Lee 002213-0481.2.3 History of TransformationThe idea of transformation was first introduced when Frederick Griffiths discovered in 1928 that a non- nonvirulent5 strain of Streptococcus Pneumoniae6 could turn virulent7 when exposed to virulent strains ofthe same species [3]. Figure 2: Griffith’s experiment and its results sGriffiths’ experiment paved a way for Avery, McCarty, and MacLeod to find out in 1944 that specialloops containing genetic information can be transferred from one bacterium to another. When cellmembrane was ruptured due to heat shock, DNA was released from heat lled Pneumoniae to heat-killednonpathonogenic strain, which showed newly acquired gene. This phenomenon was known to betransformation.5 Harmless6 Harmful7 Virulent strain of bacteria Page 8 / 36
  • 9. Chaehyun Lee 002213-0481.2.4 Uses of TransformationTransformation has a wide range of uses. One of its earliest uses is harvesting human insulin fromtransformed bacteria [4]. This is particularly useful because patients suffering from diabetes can have aready supply of insulin at hand. Since human insulin is used instead of previous sources such as sheepand cattle, allergies will rarely occur.Another notable use of transformation was made possible by the introduction of GFP, which is a proteinthat allows bioluminescence [5]. The potential of transformation was acknowledged in the ChemistryNobel Prize Award in 2008. This GFP, found in jellyfish, creates bioluminescence, and glows almostimmediately when synthesized because it does not require a substrate. GFP has potential to allow non-invasive diagnosis and protein tracing. For instance, if researchers want to know whether gene X isinvolved in the formation of blood vessels, they can link gene X to the GFP protein. If the blood vessesglow with GFP, it would be an indication that gene X is involved in blood vessel formation. Moreover,when GFP is used in eukaryotic human liver cells, researchers can detect transfected cells by using theglowing GFP [6]. Figure 3: Glowing jellyfish with GFP Page 9 / 36
  • 10. Chaehyun Lee 002213-0482.0 HypothesisToday, the widely accepted view is that most bacteria are incompetent because they are unable toabsorb plasmids directly from their surroundings. Hence, in order to artificially induce competency ofbacteria, heat shock method has to be used. The method is theorized to work as follows:First, incompetent bacteria are subjected to heat shock, which is a sudden increase in temperature.Then, HSPs8 are activated which cause pores on the membrane of bacteria to dilate or new pores to becreated. Then, as Calcium Chloride is introduced, Calcium ions (Ca2+) 9 neutralize the negative chargesfound on the cell surface of bacteria and DNA. This thereby allows a DNA molecule to adhere to thesurface of bacteria. Then, Chloride (Cl-) ions enter bacteria. This sudden influx of chloride ions creates athermal imbalance on either side of the cell membrane, which forces the plasmid DNA to enter the cellsthrough cell pores. Bacteria become competent.The most crucial step above is to make sure that Heat Shock proteins are activated because Heat ShockProteins activate reactions that eventually lead to the artificial competency of bacteria.It is very essential to find out the optimum temperature because this will allow the greatest percentageof Heat Shock Proteins to be activated. If the temperature is too low, the heat shock proteins will not beactivated. However, if the temperature is too high, bacteria may die due to protein denaturization.Note: The exact mechanics of transformation is unknown. Thus, the above hypothesis is purelytheoretical although widely accepted.8 Heat shock proteins, which are expressed when cells are suddenly exposed to sudden increase in temperature9 Cation that allows the entrance of plasmids through cell membrane Page 10 / 36
  • 11. Chaehyun Lee 002213-0483.0 Variables3.1 Manipulated Variable: Temperature used for heat shock methodHeat shock treatment is vital in transformation of bacteria because most bacteria are unable to directlyabsorb the plasmid from their surroundings. Thus, bacteria have to experience sudden increase in heatso that their plasma membrane would open and allow plasmids to be absorbed.To measure the temperatures accurately, digital Logger Pro temperature probe device was used.The following is a desired list of different temperatures that will be used for heat shock. Temperature/ ℃ Expected qualitative observation 9.0 No transformation should occur because bacteria wasn’t subject to sudden increase in heat. Heat shock proteins are not expected to be activated. 30.0 Some colonies of bacteria will be transformed and appear on LBA 10plate due to change in temperature. However, the number of colonies is not expected to be as many as that of a higher temperature (42. 0℃). 42.0 Majority of bacteria will be transformed because they will effectively absorb plasmid due to change in heat. 51.0 No transformation should occur because most proteins will be denatured.Table 1: Range of temperature for heat shock10 Luria Broth- Ampicillin. Non-transformed bacteria cannot survive in LBA conditions. Page 11 / 36
  • 12. Chaehyun Lee 002213-0483.2 Responding Variable: The CFUs11 formed on the LBA plate at the end of experiment,represented as the transformation efficiency rate.Ampicillin contains antibiotic properties. Thus, when bacteria that have gone through transformationare plated on a LB-ampicillin plate, only the ones that are resistant to ampicillin can survive and formcolonies. However, bacteria that are not transformed have no resistance to ampicillin and thereby can’tsurvive on LBA plate.The surviving bacteria colonies will be counted and recorded. Transformation efficiency rate will bemeasured using the following calculations.Step 1: Find the total mass of plasmid in fraction ୤୰ୟୡ୲୧୭୬ ୭୤ ୱ୳ୱ୮ୣ୬ୱ୧୭୬ ୮୳୲ ୭୬ ୮୪ୟ୲ୣMass of plasmid used x ୘୭୲ୟ୪ ୴୭୪୳୫ୣ ୭୤ ୱ୳ୱ୮ୣ୬ୱ୧୭୬Step 2: Calculate transformation efficiency ୒୳୫ୠୣ୰ ୭୤ ୡ୭୪୭୬୧ୣୱTransformation efficiency= ୘୭୲ୟ୪ ୫ୟୱୱ ୭୤ ୮୪ୟୱ୫୧ୢ ୧୬ ୤୰ୟୡ୲୧୭୬11 Colony Forming Units (CFU) is a measure of viable bacterial numbers. Page 12 / 36
  • 13. Chaehyun Lee 002213-0483.3 Controlled Variables Type of bacteria: Escherichia Coli DH5 ૒Modified E. Coli will be used throughout the experiment. This modified stain of E. Coli is relatively safeand is not pathogenic. Contamination will be prevented by tightly sealing the plate and the bottle thatcontains E-coli. Contamination can also be reduced by not talking (opening mouth to let out bacteria)while performing transformation. Type of Plasmid: PGREEN12Plasmid P-Green will be used throughout the entire experiment. Antibiotic: AmpicillinAmpicillin will be used to test the transformation efficiency of bacteria. Ampicillin will be poured in LB tocreate LB-ampicillin plate. Ampicillin also prevents contamination from external source because bacteriathat are not competent to ampicillin cannot survive. Incubation methodTo ensure that bacterial growth is systematic throughout the entire experiment, incubation period andtemperature will be kept constant. Period is for 24 hours and temperature is 37 ℃.12 See Appendix 2 Page 13 / 36
  • 14. Chaehyun Lee 002213-0484.0 Methodology4.1 General Overview of Methodology ethodology 9℃ Page 14 / 36
  • 15. Chaehyun Lee 002213-0484.2 Preparation of Ampicillin-LB agar (LBA) plate 1. Melt 500ml of LB agar13 in a microwave. 2. 0.300mg of Ampicillin is added to LB agar when the temperature of LB is dropped down to 57.0 ℃. 3. Mixture is mixed thoroughly and poured into petri dishes. 4. The dishes are left at room temperature (37℃) for 2 minutes until LB is solidified. 5. LB plates are turned upside-down. *This is to prevent water vapor from dropping on LB agar. 6. LB plates are stored in a refrigerator.4.3 Preparation of Competent Bacteria 1. Four 1.5ml microcentrifuge tubes are prepared and are labeled: 30 , 42 , 51 , 9 . ℃ ℃ ℃ ℃ 2. 250ul of ice-cold Calcium Chloride is placed in each microcentrifuge tube. 3. Using a sterilized loop, a single colony of E.coli culture is placed in each microcentrifuge. 4. Each mircrocentrifuge tube is stirred using vortex. 5. All microcentrifuge tubes are placed on ice for 5 minutes. 6. 5ul of plasmid pGREEN is added to each microcentrifuge by using a micropipette. 7. Microcentrifuge tubes are returned to ice for at least 15 minutes. Bacteria are ready to undergo heat shock.4.4 Transformation (Heat shock procedure)4.4.1 At temperatures of 30 , 42 , 51 ℃ ℃ ℃ 1. Waterbaths at temperatures 30 , 42 , 51 ℃ ℃ ℃ are prepared. 2. Each microcentrifuge is placed in a float and dropped into corresponding waterbath (Microcentrifuge labeled 30 ℃ is placed in 30 ℃ waterbath). A stopwatch is started immediately. 3. After 90 seconds, all microcentrifuges and floats from each water bath are removed at the same time. They are returned to ice for 1 minute.13 See Appendix 1 for preparation of method Page 15 / 36
  • 16. Chaehyun Lee 002213-048 4. 250ul of LB is added to each microcentrifuge and are left at room temperature for 15 minutes. 5. 100ul of transformed cells are removed from one microcentrifuge by using micropipette and are spread evenly on the surface of LB Ampicillin plate. This step is done for all three microcentrifuges (30 , 42 , 51 ). ℃ ℃ ℃ 6. Petri dishes are turned upside-down and incubated at 37 . ℃4.4.2 At temperature 4 ℃ 1. The temperature of a refrigerator is set at 4 . ℃ 2. Microcentrifuge labeled 4 ℃ is placed in the refrigerator. A stopwatch is started immediately. 3. After 90 seconds, the microcentrifuge is removed from the refrigerator and is placed on ice for 1 minute. 4. 250ul of LB is added to microcentrifuge and is left at room temperature for 15 minutes. 5. 100ul of transformed cells are removed from the microcentrifuge by using micropipette and are spread evenly on the surface of LB Ampicillin plate. 6. Petri dish is turned upside-down and incubated at 37 . ℃ Diagram 1: Example of heat shock transformation method Page 16 / 36
  • 17. Chaehyun Lee 002213-0484.4.3 Collection of Results 1. After incubation for one night, the petri dishes are removed from the incubator. 2. Petri dishes are placed on top of a black paper. CFU Figure 4: Inverted petri dish showing e-coli colony 3. Number of bacterial colonies is counted and noted. 4. This experiment is duplicated to reduce experimental error.4.4-4 Observation of glowing pGREEN bacteria 1. After incubation, petri dishes that contain transformed bacteria are brought into a dark room. There should be no presence of light. 2. Ultra violet light is shined directly above the petri dishes. UV light CFU CFU Figure 5: Glowing E-coli colonies under UV light 3. Glowing bacterial colonies are observed. Page 17 / 36
  • 18. Chaehyun Lee 002213-048 5.0 Data Collection 5.1 Raw Experimental Data 5.1.1 Qualitative DataControl Figure 6: Positive control (left) and negative control (right) [Left] E-coli colonies thrived in LB plate. [Right] There is no presence of any colony in LBA plate. Figure 7: Trial 1 CFU count of 9℃ (left) and Trial 2 CFU count of 9℃ (right) At 9℃ [Left] There are no observable colonies present on the plate. [Right] There are no observable colonies present on the plate. Figure 8: Trial 1 CFU count of 30℃ (left) and Trial 2 CFU count of 30℃ (right)At 30 ℃ CFU [Left] It is possible to observe the production of 3 colonies. At 9℃ [Right] There are no observable colonies present on the plate.℃ CFU Figure 9: Trial 1 CFU count of 42℃ (left) and Trial 2 CFU count of 42℃ (right)At 42℃ [Left] It is possible to observe the production of 6 colonies. [Right] It is possible to observe the production of 4 colonies.At 51 ℃ Figure 10: Trial 1 CFU count of 51℃ (left) and Trial 2 CFU count of 51℃ (right) [Left] There are no observable colonies present on the plate. [Right] There are no observable colonies present on the plate. Page 18 / 36
  • 19. 5.1.2 Quantitative Data Temperature, Number of Number of Average ℃ CFUs in Trial CFUs in Trial number of 1 2 CFUs/ CFU 9 - (a) - - ଷ 30 3 - ଶ = 1.5 ଵ଴ 42 6 4 =5 ଶ 51 - - -Table 2: Average CFU at different temperatures(a)= No CFU is found
  • 20. 5.2 Data Processing 5.2.1 Calculation of Transformation EfficiencyBefore calculating the transformation efficiency, the total mass of plasmid must first be found. The totalmass of plasmid can be calculated by using the formula below: ி௥௔௖௧௜௢௡ ௢௙ ௦௨௦௣௘௡௦௜௢௡ ௣௨௧ ௢௡ ௣௟௔௧௘Total mass of plasmid= (Total mass of plasmid) X ்௢௧௔௟ ௩௢௟௨௠௘ ௢௙ ௦௨௦௣௘௡௦௜௢௡Mass of plasmid used = Concentration of plasmid X Volume of plasmid solution used = 20ng/µl X 5µl = 100ng = 0.1µgFraction of suspension put on plate: 100µlTotal volume of suspension: 500µl ଵ଴଴ ହ଴଴ = noitcarf ni dimsalp fo ssam latoT X )1.0(20.0 = µg:woleb alumrof eht gn isu detaluclac eb won nac ycneiciffe noitamrofsnarT ே௨௠௕௘௥ ௢௙ ௖௢௟௢௡௜௘௦Transformation efficiency= ்௢௧௔௟ ௠௔௦௦ ௢௙ ௣௟௔௦௠௜ௗ ௜௡ ௙௥௔௖௧௜௢௡ Temperature, ℃ Number of Number of Average Transformation CFUs in Trial 1 CFUs in Trial 2 number of efficiency CFUs/ CFU (colonies/ µg ) 9 - (a) - - ଴ =0 ଴.଴ଶ ଷ ଷ 30 3 - ଶ = 1.5 = 150 ଴.଴ଶ ଵ଴ ଵ଴ 42 6 4 =5 = 500 ଶ ଴.଴ଶ 51 - - - ଴ =0 ଴.଴ଶTable 3: Transformation efficiencies for Trials 1 and 2 at different temperatures(a) = No CFUs found
  • 21. Transformation Efficiency/ colonies μg-1 against Temperature/ oC 600 500 Transformation Efficiency/ colonies μg-1 400 300 Transformation Efficiency 200 100 0 9 30 42 51 Temperature/ oCGraph 1: Shows transformation efficiency against temperature
  • 22. 6.0 Statistical Analysis6.1 ANOVA TestIn order to find out which of the temperatures done in this experiment is significant, an ANOVA test [7](Analysis of Variance Test) is done. Raw data collected is used for this statistical test.The ANOVA test will determine whether null or alternate hypothesis will be accepted.Null hypothesis (H0): No significant difference among different temperaturesAlternate hypothesis (HA): There is a significant difference among different temperatures.Null hypothesis is accepted if F ratio < F critical.Alternate hypothesis is accepted if F ratio > F critical.Source of Sum of Degree of Mean F Ratio Critical F P Value 2Variation squares freedom squares (s ) (df)Between 33.3 3 11.1 6.81 6.59 (computerGroups generated)Within 6.5 4 1.63GroupsTotal 39.8 7Table 4: Results of the ANOVA Test6.81(F Ratio) > 6.59 (F Critical) = Null hypothesis rejected and alternate hypothesis accepted.There is a significant difference among different temperatures.Heat shock treatment does have impact on transformation efficiency.
  • 23. Chaehyun Lee 002213-0486.2 Turkey’s HSD TestA significant F ratio only shows that the aggregate difference among the means of the several samples issignificantly greater than zero. It does not show whether any particular sample mean significantly differsfrom any particular other. Thus, Turkey’s HSD test will be done by comparing all possible pairs of groupsto determine which pair is greater than the critical value.The critical value of Turkey’s HSD is found to be: ୑ୗ౭ Critical Value = 3.62 ට ୬ ଵ.଺ଷ = 3.62 ට ଶ = 3.27 Group combination Mean difference Critical value Implication Temperature / ℃ 9 30 1.5 – 0 = 1.5 3.27 No Significant difference 9 42 5–0=5 3.27 Significant difference 9 51 0–0=0 3.27 No Significant difference 30 42 5 – 1.5 = 3.5 3.27 Significant difference 30 51 1.5 – 0 = 1.5 3.27 No Significant difference 42 51 5–0=5 3.27 Significant differenceTable 5: Comparison of mean difference and critical valueThere is a significant difference in the mean number of CFUs between two different temperatures forthe below three sets. The following pairs have a mean difference greater than critical value.There is a significant difference in the mean number of CFUs between: o 9℃ , 42℃ o 30℃ , 42℃ o 42℃ , 51℃Notice that all pairs involve the temperature of 42℃. Page 23 / 36
  • 24. Chaehyun Lee 002213-0487.0 Evaluation7.1 Explanation of ResultsFrom the results, transformation efficiency at 9℃ and 51℃ is zero.Possible reasons for the result can be as following: a. Cells were unable to take in plasmid and were therefore unable to synthesize beta lactamase which hydrolyzes ampicillin. b. Proteins in cells were denatured due to high temperature.At 9℃, there was no sudden change in temperature which means that bacteria failed to go throughheat shock. Thus, HSPs were not activated. Pores on cell membrane were not stimulated and made itimpossible for bacteria to take in plasmids. Therefore, bacteria at 9℃ were incompetent and failed tosurvive in antibacterial (LB- ampicillin) environment.Another possible explanation is that the prolonged exposure to cold had damaged the bacteria andthereby slowed down their growth. E.Coli hibernates when exposed to freezing temperatures. Hence,when they were exposed to 9℃ conditions for an extended period, they might have started hibernation.Hibernation prevents molecules from moving in and out of the bacteria. This results in no plasmiduptake and therefore no antibacterial resistance.At 51°C, proteins in cells were denatured due to high heat. Since important enzymes were unable tomaintain bacteria’s active site, reactions could no longer be catalyzed and bacteria were killed. HSPs,which could have reformed the denatured protein, were also denatured. Plasmids are also veryvulnerable to high temperature. Thus, when they were subjected to 51°C, they denatured rapidly. Thehigh temperature had denatured both plasmids and bacterial proteins, which made it highly impossiblefor transformation to occur, resulting in zero transformation efficiency.At 30°C, transformed bacteria colonies were only present on one plate (trial 1). Results fromTurkey’s HSD test shows that this temperature doesn’t reach the critical value. Possible explanation forthe low efficiency is that the temperature increase was not high enough to activate HSP. It is known that Page 24 / 36
  • 25. Chaehyun Lee 002213-048HSPs are activated when cells undergoes stress, such as exposure to high temperature. However,temperature at 30°C was not high enough to give stress to cells. Thus, HSPs remained inactivated andallowed only small amount of plasmids to be taken in.At 42°C, there was the highest transformation efficiency. 42°C was high enough to provide suddenincrease in temperature and activate HSPs. Thus, the most number of pores were created whichresulted in greater influx of plasmids and Cl- ions. Plasmids gave antibacterial resistance to E-coli colonyand allowed the most number of them to survive in LB-ampicillin conditions.Therefore, heat shock is most effective at 42°C because this temperature is high enough to create poresand keep it open for plasmids to enter but short enough to prevent denature of plasmids and bacterialproteins.7.2 Uncertainties and LimitationsThe amount of bacteria used for each trial varied because it was impossible to take out the exactly sameamount of bacteria each time by using the loop. Although careful attention was paid to take out thesame size of colony each time, it is uncertain how much bacteria were transferred. This could have hadsignificant impact on the results because the greater amount of bacteria would result in higher chancesfor transformation and vice versa.Counting the number of colonies couldn’t be accurate because there is a chance that two colonies thatare placed next to each other are merged and are mistaken as one. This would lower the transformationefficiency count because less number of colonies would be counted. Also, due to the limitations of thesight of naked eyes, smaller sized colonies could have been overlooked or mistaken as air bubbles.There is a possibility of contamination that could have possibly introduced new strands of bacteria thatare resilient to the antibiotics. Although it is assumed that ampicillin in the agar would kill anycontaminants, some bacteria may be resilient.The final temperature cannot be conclusively said to be the optimum temperature because of lack oftrials and replicates. Because of the lack of plasmid pGREEN, this experiment was limited to duplicationrather that the preferred triplication. Since transformation has a high margin of error, two trials cannotbe enough to conclusively say that the results are accurate and precise. Page 25 / 36
  • 26. Chaehyun Lee 002213-048The range of temperatures used was very limited. In this experiment, only four different temperatureswere used due to lack of laboratory facilities. These four temperatures are not enough to draw a preciseconclusion because there is a wide range between two temperatures, such as 30°C and 42°C.7.3 Ways to ImproveIn order to control the amount of bacteria that are used in each trial, a more effective equipment shouldbe used such as rotating incubator. This equipment can help make sure that equal amounts of bacteriago into each respective microcentrifuge tube by preventing the clumping of bacteria.In the final steps of the experiment, electronic bacterial colony counter should be used to detect thenumber of colonies with higher accuracy. Counting the number of colonies using naked eye (manualdetection) is less effective because smaller colonies, that are hard to see with naked eyes, may beoverlooked. Thus, an electronic bacterial colony counter will enhance the accuracy of the experiment byreducing the human error.A wider range of temperatures should be experimented to accurately determine the optimumtemperature. Since there is a big range between two temperatures, for example 30°C and 42°C, moretemperatures should be used to draw a more precise conclusion.To further enhance the precision and accuracy of the experiment, more plates should be tested per trial.During this experiment, only two plates were used. This is not suitable for transformation becausetransformation technique has a high margin of error. Thus, more plates should be done and more trialsshould be carried out (triplicate) and the average should be computed to get a more accurate data.7.4 Further InvestigationGreen Fluorescent Protein (GFP) used during transformation should be further applied to research as areporter molecule. A reporter molecule is one protein, such as GFP, linked to the protein that is subjectto study. By locating the protein with the reporter molecule, it becomes possible to follow what theprotein is doing. For an instance, if one wanted to know whether gene X was involved in the formationof blood vessels, one can link gene X to the GFP gene. Then, the cells would make a protein that was Xplus GFP, resulting in fusion protein. Thus, if the blood vessel began to glow with GFP, it would be a hintthat protein X was involved in blood vessel formation. Page 26 / 36
  • 27. Chaehyun Lee 002213-048GFP has a great potential for the future of microbiology and other medical fields because it can also beapplied to eukaryotic cells such as human liver cells. Transfection will allow researchers to easily detecttransfected cells by using the glow of GFP. This method becomes highly useful when studying cancer andneurobiology.To increase transformation efficiency, other factors that might impact transformation should also bestudied. An example would be trying out different concentrations of Calcium chloride. Ca2+ ionsproduced from CaCl2 are crucial factors in heat shock process. It is important to locate the effects ofincreasing or decreasing the concentration of Calcium chlorides because it will allow the experiments tonot waste any amount of plasmid and improve the transformation protocol.The impact of duration of heat shock on transformation efficiency should also be investigated. A longerheat shock but at a lower temperature might produce more number of colonies than a brief heat shockat a higher temperature. This is because the longer heat shock duration increases the chance ofexogenous plasmid entering the bacteria. This might lead to an alternate, even more effective,transformation treatment. Page 27 / 36
  • 28. Chaehyun Lee 002213-0488.0 ConclusionBased on the evidence obtained during this investigation, it can be concluded that the optimumtemperature for heat shock method is 42℃. This temperature is high enough to activate most heatshock proteins and low enough to prevent proteins from denaturing. At 42℃, activated HSPs enlargedand created pores on the bacterial cell membrane and significantly increased the uptake of plasmids.Hence, the highest transformation efficiency was achieved.At 30℃, transformation efficiency was not as high as that of 42℃ because 30℃ was not high enough toinduce most HSPs to be activated. Nonetheless, 30℃ condition was more efficient for heat shock thanother extremely low and extremely high temperatures such as 9℃ and 51℃.At lower temperature (9℃) heat shock proteins were not fully activated because there was no suddenincrease in temperature. In contrast, at higher temperature (51℃), bacteria died due to plasmiddenaturalization. In this condition, transformation may occur because HSPs are still activated but thetransformation efficiency cannot be high because the temperature is too high for bacteria too survive. Page 28 / 36
  • 29. Chaehyun Lee 002213-0489.0 AppendixAppendix 1- Preparation of Luria Broth agarReagents:2. 5 g – Sodium Chloride3. 5 g – Tryptone4. 7.5 g – Bacteriological Agar5. 500 ml – WaterMethodology:The reagents above are each measured using an electronic weighing machine and are all placed in abeaker. 500ml of distilled water is added to the beaker. Heat the beaker using a Bunsen burnerwhile stirring the contents with a glass rod. When all reagents are homogenized, place them in apressure bottle and sterilize using autoclave machine. When still in hot liquid state, pour the LB agarinto petri dishes. Each petri dish should be about half-filled. Agar is left to cool. Page 29 / 36
  • 30. Chaehyun Lee 002213-048Appendix 2- ANOVA testNull hypothesis H0 = u1= u2= u3= u4 means that the change in temperature doesn’t cause E-coli to havegreater transformation efficiency.Alternate hypothesis is defined as HA= one or more means are different. This means that there is asignificant difference between groups. Thus, it can be concluded that the heat shock treatment doeshave impact on transformation efficiency.All significances for the following test will be at 5% of 0.05.Assumption for the ANOVA test: 1. Observations are independent of each other. Colony formation for one result does not influence the colony formation for another result. 2. The observations in each group conform to normal distribution. 3. Variances of all groups are homogeneous (equal).The ANOVA test can be summarized as the table below:Source of Sum of Degree of Mean squares F Ratio Critical F P ValueVariation squares freedom (s2) (df)Between SSb (k-1) MSୠ Fk-1, N-k ( computer ଶ ୗୗ୵ MSୠ = MS୵Groups ୒ି୩ generated)Within SSw (N-k) ୗୗ୵Groups MS୵ = ଶ ୒ି୩Total SSt (N-1)Table 6: Summary of ANOVA calculations Page 30 / 36
  • 31. Chaehyun Lee 002213-048 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 ૛ ‫܊܁ۻ‬ Mean squares (variance) between groups ‫ܟ܁ۻ‬ ૛ Mean squares (variance) within groupTable 7: Legend for ANOVA testTemperature/ ℃ 9 30 42 51 2 2 2 2 X X X X X X X X Result 1 - - 3 9 6 36 - - Result 2 - - - - 4 16 - - 0 0 3 9 10 52 0 0 Σx ΣΣx2Table 8: Calculation for ANOVA(-)= No CFUs foundTotal summation Σx = 13Summation for squared ΣΣx2 = 61 ୗ୳୫୫ୟ୲୧୭୬ ୭୤ ଽ℃)మ ୗ୳୫୫ୟ୲୧୭୬ ୭୤ ହଵ℃)మ (ஊ୶ )మSSb = ቂ ቃ…… +ቂ ቃ – ୒୳୫ୠୣ୰ ୭୤ ୲୰୧ୟ୪ୱ ୒୳୫ୠୣ୰ ୭୤ ୲୰୧ୟ୪ୱ ୘୭୲ୟ୪ ୬୳୫ୠୣ୰ ୭୤ ୲୰୧ୟ୪ୱ ଴మ ଷమ ଵ଴మ ଴మ ଵଷమ =ቂ + + + ቃ- ଶ ଶ ଶ ଶ ଼ = [ 0 + 4.5 + 50 + 0 ] – 21.1 = 54.5 – 21.2 = 33.3 Page 31 / 36
  • 32. Chaehyun Lee 002213-048 ୗ୳୫୫ୟ୲୧୭୬ ୭୤ ଽ℃)మ ୗ୳୫୫ୟ୲୧୭୬ ୭୤ ହଵ℃)మSSw can be found by ΣΣx2 – ቂ ……+ ቃ ୒୳୫ୠୣ୰ ୭୤ ୲୰୧ୟ୪ୱ ୒୳୫ୠୣ୰ ୭୤ ୲୰୧ୟ୪ୱ = 61 – 54.5 = 6.5The total sum of squares (SSt) is SSw + SSb = 6.5 + 33.3 = 39.8Calculating the mean squares/ variance ୗୗౘ ୗୗ౭MSୠ = MS୵ = ୢ୤ౘ ୢ୤౭ ଷଷ.ଷ ଺.ହ = = ଷ ସ = 11.1 = 1.63 ୑ୗౘThe F ratio for this experiment is = ୑ୗ౭ ଵଵ.ଵ = ଵ.଺ଷ = 6.81F critical of F 3,4 = 6.59 (refer to the table below) Page 32 / 36
  • 33. Chaehyun Lee 002213-048 Figure 11: Percentiles of F Distribution Page 33 / 36
  • 34. Chaehyun Lee 002213-048Appendix 3- Turkey’s Honestly Significant Difference (HSD) ெௌೢ HSD = q (α, k, N-k) ට ௡The value of α= 0.05 (represents the significant level) k = 4 (represents the total number of groups) N-k = 4 (Total number of results – total number of groups) q = the value based on α, k, N-k = 3.62 Page 34 / 36
  • 35. Chaehyun Lee 002213-048Appendix 4- Graphical interpretation of Turkey’s HSD Graph of mean difference above critical value 6 5 4 Mean difference 3 2 1 0 9 – 30 9 -42 9 – 51 30 – 42 30 – 51 42 - 51 Temperatuer pair/ C° Mean difference Critical valueGraph 2: Shows temperature pairs that are greater than the critical value Page 35 / 36
  • 36. Chaehyun Lee 002213-04810.0 Bibliography[1] "Escherichia Coli." Medical News Today. MNT, 08 June 2011. Web. 1 Jan 2012.<http://www.medicalnewstoday.com/articles/68511.php>.[2] "Gram-negative." Howard Hughes Medical Institute. HHMI, 2011. Web. 1 Jan 2012.<http://www.hhmi.org/biointeractive/Antibiotics_Attack/bb_2.html>.[3] "Transformation." Science & Technology Education Program. Lawrence Livermore NationalLaboratory, n.d. Web. 1 Jan 2012. <http://education.llnl.gov/bep/science/10/tLect.html>.[4] Rapoza, Maria. Transformations. Burlington: Carolina Biological Supply Company,[5] Zimmer, Marc. Green Fluorescent Protein. N.p., 15 June 2011. Web. 1 Jan 2012.<http://gfp.conncoll.edu/>.[6] Gleiberman, Anatoli. "Expression of GFP in adult liver." . National Library of Medicine, 2009. Web. 1Jan 2012.[7] "Analysis of Variance Between Groups." Tools for Science. MailTo, n.d. Web. 16 Jan 2012.<http://www.physics.csbsju.edu/stats/anova.html>. Page 36 / 36