Purification of Thermus Aquaticus DNA Polymerase - Lab Report Example

Introduction: The objective of this practical was to express and purify the thermostable DNA polymerase from Thermophilus aqauticus (Taq polymerase) using this Taq polymerase to amplify target gene in order to identify the unknown organism. The polymerase chain reaction (PCR) is a tool that is used to amplify DNA, where at every thermal cycle a complementary DNA strand is synthesised. (Mullis et al 1986) With the repeated cycles of complementary DNA strand synthesis, there is an accumulation of new synthesised DNA strands that could be modelled either to rapidly synthesis many strand of DNA or synthesis a few strands. (Ruijter et al 2009, Rutledge and Stewart 2010) The PCR kit is a widely used technique in the amplification of DNA strands and as a result it has many applications in various of synthesising complementary DNA strands to make it possible to amplify a minutes DNA sample so that it could be detected if when it is very small amount of DNA in any given sample. The Taq polymerase first isolated from Thermophilus aquaticus is preferred because it could withstand high temperatures used in the PCR amplification of DNA strands. The PCR amplification of DNA strands takes place in several cycle of heating and cooling the thermal cycles stimulate the Taq polymerase primers to bind to the complementary DNA strands of the newly amplified DNA strands. The amplified DNA strands are then run in a SDS PAGE gel electrophoresis where the strands of DNA are separated according to there molecular weight, these separated DNA strands could then be visualized directly by staining with ethidium bromide. (Erlich 1989) Techniques involved Transformation and analysis of transformation efficiency The process of transformation bacterial cells to absorb DNA strands of another organism, the process to be used in this experiment is chemical transformation process. Where by the bacterial cells were treated with divalent metal ions to make cells competent to absorb DNA. Expression and Purification of Taq Polymerase The Taq DNA polymerase is used in PCR as a primer to bind to the DNA in the sample so that the complementary synthesis of new DNA strands takes place. (Engelke et al 1990) In the presence of a DNA template in the sample, the Taq polymerase primer terminus and Mg++ catalyzes the incorporation of dNTPs into sample DNA because the Taq polymerase contains a polymerization dependent 5'-3' exonuclease activity that initiated elongation process of DNA synthesis. (Grimm and Arbuthnot 1995) The expression of the Taq polymerase gene is under the control of the lac promoter system. This means that if the Taq polymerase gene has to be expressed galactose has to be present in media. In this experiment the non-hydrolysable galactose analogue IPTG was used to induce the cells to express protein. The cells cannot metabolize IPTG, therefore, the cells will continuously keep expressing the protein. (Saiki 1985) SDS-PolyAcrylamide Gel Electrophoresis The SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is used to separate DNA according its molecular weight. The DNA double strand is separated into single strands in the SDS-loading buffer where the detergent Sodium Dodecyl Sulphate (SDS) bind to the single stranded DNA, keeping it unfolded and giving it a uniform mass to charge ratio when the gel is run. Using SDS-PAGE you will be able to show the DNA present in your sample. This will allow you to show the purification of the Taq polymerase through the steps of expression and purification. You will need to cast an SDS-PAGE gel. Each gel will allow you to run a total of 10 samples so gel can be shared between two. (Grimm and Arbuthnot 1995) Used a prepared Taq polymerase from fellow bench members and the provided Taq polymerase by staff members and the samples were run in the thermal cycler by the staff. Material and Methods Bacterial strains Serratia marcescens NCIMB 1377 Pseudomonas fluorescens NCIMB 8194 Micrococcus luteus NCIMB 196 Bacillus subtilis sub sp niger NCIMB 8649 Bacillus cereus var mycoides NCIMB 926 Proteins Hexokinase, Phosphofructokinase, Triose Phosphate Isomerase, Glyceraldehyde phosphate dehydrogenase, Phosphoglycerate kinase, Pyruvate kinase Transformation procedure Take aliquot of competent cells on ice and add 1µl of plasmid DNA (10ng/µl) Incubate on ice for 15 minutes. Heat shock at 42 ºC for 90 seconds. Incubate on ice for 2 minutes. Add 900 µl of LB and incubate at 37 ºC for 1 hour. Mix the cells by gently pipetting up and down. Pipette 100 µl of cells onto a LB-amp plate and spread across the surface of the plate and spin the cells briefly in a microfuge for 30 seconds to pellet all remaining cells. Decant off the supernatant leaving a small drop behind. Resuspend the pellet by gently pipetting the remaining solution up and down. Pipette the remaining solution onto the second plate and spread across the surface. Incubate the plates overnight at 37 ºC. After incubating the plates overnight the cells that are transformed will grow into colonies. By counting the number of colonies that have grown can calculate the transformation efficiency. Take pictures of both the plates and count the number of cells on each plate. Transformation Efficiency = Total of cells on agar plate Amount of DNA spread on agar plate Taq Polymerase purification Inoculate a 1 ml overnight culture of LB-amp with a single colony from an E. coli Taq plate and grow overnight at 37 ºC in a shaking incubator. Add ampicillin to a final concentration of 100mg/litre to the 50 ml flask of sterile LB (stock is 100 mg/ml) after autoclaving. Add the overnight cultured cells to flask of LB-Amp. Incubate the culture at 37 ºC shaker for approximately 3 hours. Take out 125 ml sample and place in a 1.5 ml centrifuge tube. Spin at 13k rpm for 1 minute. Decant the supernatant add 20 ml H20 and 20 ml SDS loading buffer. Incubate at 95 ºC for 4 min to make a sample for SDS-PAGE and label it as ‘un-induced’ (S1) and repeat this step to make (S2) induced. Add IPTG to 50ml flask to a final concentration of 0.4 mM and incubate for 3 hours. Pour the rest of the cells into a 50ml Falcon tube and centrifuge for 10 minutes to pellet cells. Carefully decant off the supernatant with a pipettor Label the 50ml tube and freeze overnight and Place 2 samples for SDS-PAGE in the fridge overnight for storage. Cell lysis and Taq Polymerase purification Resuspend the pellet by carefully pipetting up and down using the chilled 0.5 ml of Buffer A with lysozyme (ice cold). Transfer the resuspended cells back to the 2ml centrifuge tube Incubate at room temperature for 30 minutes to allow the lysozyme to digest the cell wall of the E. coli. Add 0.5 ml Buffer B and incubate at 80 °C for 15 minutes, chill on ice for 10 minutes, and then spin at 13k rpm in a microfuge for 15 minutes. Balance the tube with another tube. Transfer the supernatant to a fresh 1.5ml tube and keep on ice. Take a 20 ml sample into a fresh 1.5ml centrifuge tube and add 20 ml of SDS-loading buffer, heat at 95 ºC for 4 min. Label ‘heat-treated’ (S3) Back to the lysed cell solution, the total volume of the solution has to be determined in order to calculate the volume of ammonium sulphate to add to the solution. Weigh the volume of empty 1.5ml tube on the balance and mark it zero then place tube with the sample on. The weight gives the volume. Record this value and add pulverized ammonium sulphate slowly to a final concentration of 0.164 g/ml (30% saturation). Mix well to ensure it is fully dissolved and incubate on ice for 30 minutes. Pellet the precipitate by spinning in a microfuge at 13k rpm for 15 minutes. Transfer the supernatant to a fresh tube on ice, add ammonium sulphate to a final concentration of 0.181 g/ml (60% saturation) using the same method as before but divide the weight by 1.2 to allow for the increase in density caused by the ammonium sulphate already present. Mix and ensure fully dissolved as before. Incubate for 15 minutes on ice. Pellet the precipitate in the microfuge at 13k rpm for 15 minutes. Resuspend in 0.5 ml Buffer C take a 20 ml sample into a fresh 1.5ml centrifuge tube and add 20 ml of SDS-loading buffer. Heat at 95 ºC for 4 min and label as ‘final protein’ (S4) carefully label the final Taq polymerase and store in the freezer. SDS PAGE gel preparation Bio-Rad Mini-PROTEAN 3 Cell, Acrylamide/bis-acrylamide (30% w/v/0.8% w/v) 1.5M Tris/HCl, pH8.8 (resolving gel stock) 0.5M Tris/HCl, pH6.8 (stacking gel stock) TEMED, SDS (10% w/v) Ammonium persulphate (APS; 25% w/v) Running buffer [0.025M Tris/0.192M glycine/0.1% (w/v) SDS, pH8.3] Pre-stained molecular markers (PageRuler) Coomassie Blue Stain Solution. Taq polymerase purification analysis by SDS-PAGE electrophoresis Loading Sample The loading sample is prepared by heating all samples in a hot water bath for 2 minutes and then cools them down to room temperature. Load each of the samples into the wells slowly to allow them to settle evenly on the bottom of the well. Load 5µl the pre-stained markers to Lane 1 to control the migration of proteins during SDS PAGE gel electrophoresis. To each lane load 20 µl of respective sample as follows; un-induced to lane 2 sample (S1), induced sample to Lane 3 (S2), heat-treated sample to Lane 4 (S3) and final protein sample to Lane 5 (S4). Set SDS-PAGE gel electrophoresis at 200 volts constant is and run time is about 45 minutes until the ethidium bromide dye-front has migrated to the bottom of gel. Stain and De-stain gel Pour off and decant the running buffer before opening the cams to avoid spilling the buffer and remove the gels from the Gel Cassette Sandwich then transferred to a staining tray and submerged with 20ml Coomasie stain solution for 15 minutes with gently shaking. Decant the stain back into the bottle, rinse the gel with water first, and then add Destain (to cover the gel). Roll up a small piece of tissue and place it in the tray with the gel until the protein bands are seen clearly. Use a camera or mobile phone to take a photo of the gel. PCR-1: Control reactions Primers preparation: Make working solutions of 10 pmol / µl of purified primers by taking 10 ul of stock primer solution and adding 90 µl of water. Record the values after adding water for each tube Number the first and last tubes 1 and 4 and mark your initials. Tube 1 and 2 will contain a PCR reaction using the designed primers and the DNA template from the corresponding organism. Tube 3 and 4 will contain a PCR reaction with the same DNA template but a set of provided control primers. Tube 1 and 3 contained the purified Taq polymerase, whereas tube 2 and 4 contain PCR reaction with Taq polymerase provided by staff members. Add the water to the tube and mix it by pipetting it up and down. Transfer 49 µl from tube 1 to tube 2 and from tube 3 to tube 4. Using a loop pick colony and transfer some cell directly to the tube. Add 3 µl of either purified Taq OR the commercial Taq polymerase. To tubes 1 and 3 add of purified Taq polymerase, and to tubes 2 and 4 add 3 µl of the provided Taq polymerase and mixed well. The samples will then be run for you in the thermal cycler. PCR-2: Identification of the unknown organism Primers preparation: The working solution of primers is prepared the same way as in PCR 1. Starting from stock solution of 100 pMol / µl, make working solutions of 10 pmol / µl of freshly prepared primers by taking 10 ul of stock primer solution and adding 90 µl of water. A solution representing a body fluid sample from extracted from the individual to identify and which contains the microorganism to be identified. Use the 2 PCR tubes to set up the reactions by labelling and numbering the tubes. To tube 1 add freshly prepared Taq polymerase and tube 2 add the provided Taq polymerase. To tube 1 and 4 other tubes add 10 µl of 10x PCR buffer, 3 µl of 10 µM dNTPs, 3 µl x 4 of 10 pmol / µl Primer F, R 3 µl x 4 of 10 pmol / µl Primer and Water To Total 80 µl mix it carefully by pipetting it up and down. Transfer 40 µl from tube 1 to tube 2. Then add 7 µl of the provided Unknown Microorganism solution and add 3 µl of freshly prepared Taq to tube 1 and provided Taq polymerase to tube 2. Mixed well by flicking the tube and spinning the liquid down and use special adaptors to centrifuge PCR tubes and the samples were run in the thermal cycler by the staff members. PCR programme: Denaturation 94° C for 2mins (Denaturation 94° C for 20sec, Annealing 50°C for 20sec and Elongation 72° C for 40sec at (x 35) ) final elongation 74°C for 4mins and storage at 4° C Agarose gel electrophoresis of PCR products Method Electrophoresis buffer TAE: 40 mM Tris 20mM acetic acid; 1mM EDTA pH 7.8 Sample loading buffer 6g glycerol, 5ml 0.2M EDTA, 0.8ml saturated bromophenol blue dye, 1.0% Agarose gel 0.5g agarose in 50ml of buffer TAE, microwave to melt. Cool to 50°C. Add 50 µl ethidium bromide to give 0.5 µg/ml, PCR buffer (1x final) 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3 at 25°C Place the sample comb in the gel casting tray and set up the electrophoresis system. Pour in the gel to a depth of ~5mm and allow 20 min to set. Remove the comb. Add electrophoresis buffer (TAE) to cover the gel by approximately 0.5cm. To each PCR tube add 5 µl of DNA loading buffer and mix carefully by pipetting up and down. Add 10 µl of each sample. Record the positions of the samples in the lanes. To one lane on the gel add 5 µl of marker. Note down the size of your fragments. Run the gel at 120Volts for approximately 40 minutes or until the blue dye runs half way down the gel. View the gel under UV light. Photograph the gel to allow determination of band sizes. Results Transformation: Transformation Efficiency = Total number of cells on agar plate Amount of DNA spread on agar plate = 4/10 Transformation efficiency = 0.4 Photo of your plates; figure 1 Positive Control Taq Polymerase purification The expression of recombinant protein was induced by IPTG the Taq polymerase was cloned successively to E. coli in LB broth overnight at 37°C and added ampicillin selectively allowed the transformed E. coli to grow. Photo of the SDS gel, with legend figure 1 PCR 1 and 2 PCR 2 Pseudomonas fluorescens (1) Serratia marcescens (2) Micrococcus luteus (3) Bacillus subtilis (4) Bacillus cereus (5) The unknown microorganism was identified as Pseudomonas fluorescens as indicated in figure 2. The results indicated here was mine. Discussion Transformation The efficiency of transformation was calculated and found to be 0.5. The transformation of Taq polymerase was affected on the amount of plasmid DNA spread on the plates which in turn resulted into the amount of cells growth on the plates. The amount of cells cultured should be increased so that the efficiency of transformation would increase too. Taq Polymerase purification The DNA primers that were used were of unknown microorganism and the results obtained were matching with the standards of the known four microorganisms. The five freshly prepared Taq polymerases purity was satisfactory; more would have been done to increase the purity of the Taq polymerase through the more dilutions to completely remove contaminating DNA. The last lane indicated that the freshly purified Taq polymerase was satisfactorily purified and when it was run on a PCR it was efficient as it was identical to the standard Taq polymerase used as a control. PCR 1 and 2 The control checks’ of the PCR 1 of the first four tubes, contained the DNA primers of the known microorganisms, which were run on the SDS PAGE electrophoreses to separate the DNA the results confirmed to the standard of the five DNA primers provided by the staff members; Pseudomonas fluorescens (1) Serratia marcescens (2) Micrococcus luteus (3) Bacillus subtilis (4) Bacillus cereus (5). This indicated that the results of PCR 2 would be compared with those of PCR 1 because they were standard controls. When the results of PCR 2 were compared with those of PCR 1 they were found to be compatible, resulting in the easy identification of the unknown microorganism because the separated band in lane 5 corresponded to that of lane 1, hence a positive match was observed between lane 1 and lane 5, which positively identified the unknown microorganism as Pseudomonas fluorescens. The limitation experienced during this experiment was mainly because of the lack of standard purification process of Taq polymerase. As explained by Corless, there was always some kind of microbial DNA contamination even in commercial preparations of Taq DNA polymerases. (Corless et al 2000) During the purification process of Taq polymerase, there are chances that at the end the purified Taq polymerase would be still contaminated with either the bacterial DNA or other DNA other than that of T. aquaticus. Therefore, there is a need to observe strictly proper process of purification to guarantee a good percentage of purified Taq polymerase. The amplification of cloned DNA from Taq polymerase and transformed E. coli (Hughes et al 1994) were confirmed through restriction enzyme analysis that the contaminating DNA was not that of E. coli or T. aquaticus. (Rand and Houck 1990) This contamination is generally assumed to be due to improper purification process, during reagents addition to the enzyme or even from the sterile PCR kit. (Iversen et al 2007) The protocol needs to be lined in a way that the Taq polymerase is purified as much as possible to elimination contaminating DNA by serial dilution of Taq polymerase to increase the reliability of PCR assays for low-level bacterial DNA Reference list Corless C E, Borrow R, Guiver M, Edwards-Jones V, Kaczmarski EB (2000) Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38 pp. 1747–1752. Engelke D R, Krikos A, Brucke M E, Ginsburg D. (1990) Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Analytical Biochemistry 191 pp. 396-400. Erlich H A. (1989) PCR technology: principles and applications for DNA amplifications. Stockton Press, NY. Grimm E, Arbuthnot P. (1995) Rapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures. Nucleic Acids Research 23 pp.4518-4519. Hughes M S, Skuce RA and Beck LA, (1994) Identification of DNA sequences in Taq DNA polymerase. J Clin Microbiol 32: Iversen B G, Bo G, Hagestad K, Eriksen H M, Jacobsen T (2007) Pseudomonas aeruginosa contamination of mouth swabs. Ann Clin Microbiol Antimicrob 6 pp. 3 Grimm E, Arbuthnot P. (1995) Rapid purification of recombinant Taq DNA polymerase by freezing and high temperature thawing of bacterial expression cultures. Nucleic Acids Research 23 pp. 4518-4519. Mullis K, Scharf S, Faloona F, Saiki R, Horn G (1986) Specific enzymatic amplification of DNA in vitro Cold Spring Harb Symp Quant Biol 51(1) pp. 263–273. Rand K H and Houck H (1990) analysis of Taq polymerase containing bacterial DNA of unknown origin. Mol Cell Probes 4 pp. 445–450. Ruijter J M, Hoogaars W M, Ramakers C, Karlen Y, Bakker O (2009) Amplification efficiency analysis of quantitative PCR data. Nucleic Acids Res 37 pp. 45. Rutledge RG, Stewart D (2010) Assessing the performance capabilities of LRE-based assays for absolute quantitative real-time PCR. PLoS ONE 5 pp. 9731. Saiki RK. (1985)Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 pp. 1350-1354. Read More