Identifying the Obscure Microorganism Using the Polymerase Chain Reaction Technology - Lab Report Example

PCR 2 Author’s Name: Instructor’s Name: Course Details: Institutional Affiliation: Date of Submission: Introduction: The aim of the project was to clearly identify the unknown microorganism using the polymerase chain reaction technique. However, other techniques such as prokaryotic cell culture, transformation, gene expression, protein purification and electrophoresis were also employed in order to provide the necessary components of polymerase chain reaction. The PCR technique used in the project was developed by Karry Mullis back in 1983. The technique gave Karry Mullis and Michael Smith worldwide recognition after earning the Nobel Prize in chemistry. Consequently polymerase chain reaction has become adopted and is essential in the day to day biological research lab applications (Sambrook and Russell 2001, p. 3). The PCR technique is exquisitely sensitive in the identification or detection as well as high amplification levels of gene sequences within the shortest time possible. PCR works by amplifying the DNA or the cDNA templates to thousand or million fold in a quicker and most reliable manner. In addition, polymerase chain reaction generates enough materials for the future experimental analysis (Huang et al. 2002, p. 4). DNA in bacteria is well expressed in the plasmids. Plasmids are extra chromosomal DNA molecules and are circular in shape. Bacteria contain numerous copies of plasmids. Plasmids are able to undergo replication that is independent of the DNA of the host. The plasmid DNA is added to the competent E. coli during transformation. The transformed cells are thereafter cultured and incubated in a selective media. The growth of cells on selective media that is impregnated by antibiotics enables the identification of unknown microorganisms. The cells that contain plasmids are able to undergo growth when cultured in the selective media. The growth of the cells that contain the plasmids is attributed to the fact that they are resistant to the antibiotics that are impregnated in the selective media. The resistance to the antibiotic allows the cells to have their normal physiological process hence they are able to proliferate. However, the cells which do not contain plasmids are sensitive to the impregnated antibiotic. The sensitive cell membrane of the sensitive cells undergoes lysis and this interferes with the normal physiological processes of the sensitive cells. As a result, the growth of the sensitive cells is inhibited (Sambrook and Russell 2001, p. 3). The electrophoresis of the proportional representation of the cells that have undergone transformation results in the distribution of the cells based on their molecular weight and electrostatic forces. The molecular weight and electrostatic forces differ from one microorganism to another and this will be reflected by their distribution on electrophoresis. As a result, the comparison of the PCR product length will aid in the identification of the unknown microorganism (Smith et al. 1990, p. 38). Expression and purification: The goal of protein purification is to increase the growth of the cultures. The expression and purification of proteins was achieved by first carrying out the inoculation of the single colony of the recombinant E.coli that had the taq polymerase gene. This was followed by transforming the E.coli competent cells with the recombinant vector. Purification of the taq polymerase was there after conducted. The expression and purification of the thermostable DNA polymerase from thermophilus aquaticus (taq polymerase) was done to facilitate the identification of the unknown microorganisms. The transformation of the cells was geared towards expressing the cells that contain the vector which is a plasmid. The plasmid contains the gene encoding of the taq polymerase and will only grow in selected media. The selected media contains antibiotics impregnated in them. The impregnated antibiotic in this case was ampicillin. The cells that are resistant to ampicillin will grow in the media as opposed to those which are susceptible to ampicillin. The cells that are susceptible will be destroyed by the ampicillin due to it destroying its cells membrane as well as disrupting the normal cellular functions of the microorganisms. This means that the cells that will undergo transformation and grow on the media are those of the species to be identified. The other cells that fail to grow on the media will not be resistant to the antibiotic. The ability of the microorganisms to offer resistance to ampicillin aids the identification of e coli from other microorganisms such as Serratia marcescens, Pseudomonas fluorescens, Micrococcus luteus, Bascillus subtilis sub sp niger and Bascillus cereus var mycoides. This is because E.coli is resistant to ampicillin whereas the other microorganisms are susceptible to ampicillin (Calvin and Hanawalt 1988, p. 2798). The expression of the taq gene is controlled by the lac promoter system. The lac promoter system allows for the expression of the taq gene due to the presence of galactose. In this project, hydrolysable galactose analogue IPTG was used to induce the expression of the genes. The advantage of using the analogue is that it cannot be metabolized hence the cells continuously express the plasmids which they absorbed (Huang et al. 2002, p. 4). Inoculation: Inoculation entailed the addition of bactopeptone (yeast extract) to NaCl. This was aimed at forming 50ml of the LB media into which further 50ml of water was added. 1ml of the LB culture that contained a single e. coli colony was thereafter inoculated and cultured overnight at 7 degrees celcius in a shaking incubator (Fujii et al. 1999, p. 289). Transformation: The aim of the entire transformation process is to enhance the absorption of DNA plasmids by the cells. The absorption of the plasmids will mean that the cell become resistant to the antibiotics. Only the ells with the plasmid DNA are resistant to DNA. The resistance to DNA is unique to the DNA plasmids which are not affected by mode of action of the antibiotic. The process of transformation entails the competent e. coli being transformed with the plasmid DNA. Only a limited number of bacteria take up the plasmid DNA. The bacteria are then plated on a selective media and incubated over night. The growth of the bacteria will be controlled. This means that only the bacteria with the plasmid will grow since the selective media will be impregnated with antibiotics (ampicillin). The antibiotics will lead to destruction of the cells that do not contain the plasmid since they will not have the resistance properties (Smith and Scotland 1993, p. 12). Transformation allows for the absorption of the DNA by bacterial cells. In this case chemical transformation was employed. The bacterial microorganisms were treated with divalent metal ions. The treatment with divalent metal ions makes the bacterial microorganisms to be competent in absorbing genetic material. This means that more DNA will be absorbed by the microorganisms that have been treated with the divalent metal ions. At the end of the treatment exercise, only the competent cells exhibited the complete absorption of DNA material. In order to achieve this, the cells were incubated for 15 minutes together with plasmid DNA on ice. The incubation allows the association between surfaces of the cells and that of DNA. After the association has been established, the cells were then heat shocked at 42 degrees Celcius for a period of 90 seconds. The heat shocking resulted in the absorption of the DNA into the cell membrane of the cell. The cells were then chilled or incubated on ice for two minutes. The chilling on ice was done to allow for the stabilization of the cell membranes and 900 microlitres of LB was added to the cells. The final process in transformation entailed allowing the growth of the cells for one hour. The growth enhanced antibiotic resistance by the cell that absorbed plasmid DNA (Tarr 1995, p. 8). A second inoculation was done on the second day and it involved the addition of 100mg/ litre of ampicillin to the sterile LB stock. The culture was then grown at 7 degrees for three hours. The second inoculation was done to impregnated the plate in which the growth of the culture was done. Only the bacteria that took up the plasmids were able to grow. The growth of the bacteria is attributed to the fact that the plasmids have resistant properties to the ampicillin. Ampicillin is active on bacteria that do not contain plasmids. Ampicillin mediates its function by destroying cell walls and cell membranes of the bacteria. This interferes with the normal physiological process of the bacteria hence death ensues. This meant that the susceptible or those bacteria that did not take up the plasmids had undergone the process of cell lysis. Consequently, only the cells that had taken up plasmids were observed to form colonies (Deiffenbach and Dveksler 1995 p. 12). The transformation allowed the growth of cells to form colonies. The efficiency of the transformation process was then established by counting the number of colonies that were formed. With the number of colonies that were formed, it is easier to calculate the transformation efficiency (Deiffenbach and Dveksler 1995 p. 12). The efficiency of transformation: The efficiency of transformation is achieved by the counting of the viable bacteria. The colony forming unit is an estimate of the number of viable bacteria. Establishing the colony forming unit involves the counting of the number of colonies that were formed per milliliter when the culturing was done on the impregnated plates. Only the cells with plasmids were able to grow (Deiffenbach and Dveksler 1995 p. 12). Calculation of the efficiency transformation: 1.0 ng of control DNA (1 μL of 0.1 ng/μL, freshly diluted) was added to 100 μL of competent cells. 900 μL of SOC medium was added prior to expression. 100 μL (equivalent to 0.01 ng DNA) was diluted in 900 μL SOC and 100 μL was plated (equivalent to 0.001 ng DNA). 100 colonies were counted on the plate. 100 colonies / 0.001 ng X 1000 ng/μg = 1 x 108 T/μg. Transformation efficiency (TE) equation: TE = Colonies/µg/Dilution Colonies = the total number of colonies counted on the plate µg = the amount of DNA transformed expressed in µg Dilution = the total dilution of the DNA before plating µg= 1 ng Number of colonies=100 Dilution=10/1000 x100/1000=0.001 Transformation efficiency =colonies/ µg /dilution Transformation efficiency = 1 x 108 T/μg. Taq Polymerase Purification: The taq is a thermostable DNA dependant polymerase that was initially isolated from the prokaryotes. The taq polymerase enzyme is able to withstand protein denaturing conditions. The denaturing conditions are associated with high temperatures normally exhibited in polymerase chain reactions. The optimum temperatures for activity range between 75-80 degrees celcius. The half life lasts for 9 minutes and is at 95.5 degrees celcius. The replication of the taq is at 10 to base 3 in an interval of less than 10 seconds at temperatures of 72 degrees Celcius. The taqDNA is responsible for the catalysis of the incorporation of dNTPs into DNA. The reaction requires a DNA template which is the primer terminus and magnesium divalent cation. The taq from aquaticus has similar characteristics as that of the recombinant taqDNA that is expressed y the E.coli. The thermostablity of the taq DNA is associated with the increase in its hydrophobicity properties. In addition, its enhanced electrostatic forces as well as molecular interactions make it to be more stable since they bring about the addition of proline residues to the enzymes surface (Sambrook and Russell 2001, p. 3). Purification allows for the isolation of pure plasmids. Purification involves the use of silica-gel based spin columns being used to bind with the plasmids. The binding of the plasmids is preceded with an ethanol wash and the elution of the DNA in a small volume of tris buffer or water (Fujii et al. 1999, p. 289). SDS- PolyAcrylamide gel electrophoresis The (SDA-page) works by separating the proteins based on their molecular sizes. The SDS loading buffer enables the unfolding of the various proteins. The SDS loading buffer is used because it contains sodium dodecyl sulphate (SDS) which is a detergent with capabilities of binding proteins and keeping them in the unfolded state. The keeping them in the unfolded state is advantageous because it keeps that proteins in the unfolded state hence allowing the proteins to have a uniform mass to charge ratio when the gel is run (Calvin and Hanawalt 1988, p. 2798). As a result, the (SDA-page) was able to show the various proteins that were contained in the samples and this provided a basis of purification of the taq polymerase through the steps of expression as well as purification. The polymerase purification analysis by the SDS-page electrophoresis was achieved by first ensuring that the samples were loaded. Thereafter, all the samples in the hot block were heated at 100 degrees celcius for 2 minutes and cooled down to room temperatures (Deiffenbach and Dveksler 1995 p. 12). The loaded samples included: In Lane 1, 5µl of the pre-stained molecular markers (Page Ruler) was loaded to check migration of proteins. The Lane 2: 20 µl of the ‘un-induced’ sample (S1) was loaded. Lane 3 was loaded with 20 µl of the ‘induced’ sample (S2). The Lane 4 was loaded with 20 µl of ‘heat-treated’ sample (S3). Lastly, Lane 5 was loaded with 20 µl of the ‘final protein’ sample (S4). PCR reaction: The PCR process is divided into three stages: 1) Exponential amplification stage In this stage, every cycle results in the doubling of the product. 2) Leveling stage This is the second stage and is characterized by the slowing down of the rate of reaction. The DNA polymerase activity in this stage is slowed down. 3) The plateau stage The plateau stage is characterized by the cessation of the accumulation of the products. No more reaction takes place in this phase since all the reagents and enzymes are exhausted. The PCR reactions entail the utilization of the designed primers and taq polymerase in the identification of the unknown microorganism (Tarr 1995, p. 8). PCR 1 reactions: this set of reactions involves the setting up of control reactions that use the designed primers, corresponding microorganisms and purified taq polymerase. The first step of PCR 1 reactions is the preparation of primers. PCR 2 reactions: the PCR 2 reactions are aimed at identifying the unknown microorganisms. They involve the use of primers in the identification of the unknown microorganisms. PCR 1 and 2: The PCR is useful in the amplification of segments of DNA. The amplification process entails the use of the thermostable DNA polymerase, two nucleotide primers, deoxynecleotide triphosphates, a buffer and metal ions such as magnesium. All these components are mixed and placed in a thermo cycler. The thermo cycler provides a series of temperatures for the reaction at specific time intervals. The series of temperature adjustments and time intervals changes provides the cycle of amplification of the reaction. Each step of the amplification cycle results in the denaturing of the template. The denaturing of the template leads to the production of two nucleotide-prime single stranded DNA templates. The formation of the primers leads to the setting up of polymerization reactions in which a copy of each template is synthesized into a single strand. It is this single stranded DNA template that gives rise to the thermostable polymerase. The completion of each cycle of the amplification results in the doubling of the targeted template sequence (amplicon) (Huang et al. 2002, p. 4). The microorganisms were distributed based on their molecular weight and charge. The microorganisms in the experiment included: Serratia marcescens Pseudomonas fluorescens Micrococcus luteus Bascillus subtilis sub sp niger Bascillus cereus var mycoides The unknown micoorganism Based on the molecular weight of the microorganisms, their distribution was in lane 1, 2, 3, 4, 5 and 6 respectively. The identification of the unknown microorganism was based on the comparison of the PCR product length. This means that the unknown microorganism which is E.coli was in lane number 6. The reaction in this project was set up according to the protocol. The analysis was done using the agarose electrophoresis. Discussion: The study of E.coli has been undertaken for many years with the advent of molecular and genetic engineering. This can be attributed o exemplary characteristics that E. coli portrays as a model microorganism. Some of the exemplary features of E. coli include fast amplification, ease in isolation, high gene expression levels, its characterization and the ability to be cultured on a large scale. These aspects enable the researchers to easily use E. coli in various experiments since it has a well established genetic sequence (Sambrook and Russell 2001, p. 3). Transformation: It is evident from the experiment the transformation process was not 100% effective. The efficiency of transformation is bound to be influenced by various factors. Some of the factors that affected the transformation efficiency include the purity of the DNA, the manner in which the cells were handled and how the transformation process was carried out (Smith and Scotland 1993, p. 12). Impurities in the DNA The inefficiency caused by impurities can be well avoided by carrying out the purification reactions, ligations, endonucleous digestions and other know forms of treatments. For instance, phenol-chloroform extraction followed by precipitation by ethanol can be carried out (Smith and Scotland 1993, p. 12). The handling of the competent cells The competent cells are affected by the changes in temperature. As result, the cells must be thawed in ice and the transformation process started immediately after the cells have been thawed. In addition, the cells must be treated gently. This means that the cells have to be mixed gently by swirling and avoiding pippeting or vortexing. Finally, the transformed cells should not be thawed in ice since this will lower the transformation efficiency (Calvin and Hanawalt 1988, p. 2798). Other ways to prevent the transformation being affected is by avoiding the addition of too much ligation to the transformation. It is advisable to use less than 1 μL of a ligation. The addition of too much ligation limits the amount of transformants hence the efficiency of transformation is lowered (Sambrook and Russell 2001, p. 3). Taq Polymerase purification: Taq is not very effective due to numerous limitations and challenges. Some of the factors that influence the frequency levels include: The concentration of the divalent ions that are used in the experiment High levels of Magnesium Chloride and dNTP concentrations are bound to increase the frequency errors. This limitation can be avoided by the use of low Magnesium Chloride and dNTP concentrations (Deiffenbach and Dveksler 1995 p. 12). The number of cycles The increase in the number of cycles is bound to increase the frequency errors. The use of Taq is associated with low fidelity rates in relation to other polymerases. This limitation can be corrected by increasing its fidelity rates (Fujii et al. 1999, p. 289). The number of plates The number of plates used in the initial stages of the experiment influences the frequency. To avoid this form of error, the use of high number of plates ought to be avoided (Tarr 1995, p. 8). Proofreading The absence or presence of proofreading activity influences the frequency (Smith et al. 1990, p. 38). Proposed changes to the protocol: The concentration of the divalent metal ions to be used in the experiment should be specified to avoid transformational errors. The cycle of amplification of the reactions should also be quantified. References Calvin, N, M, and P, C, Hanawalt, 1988, Journal of Bacteriology 170: 2796-2801. Deiffenbach, C, W, and Dveksler, G, S, 1995, PCR Primer, A laboratory manual, Cold Spring Harbour: Laboratory Press. Fujii, S, Akiyama, M, Aoki, K, Sugaya, Y, Higuchi, K, Hiraoka, M, Miki, Y, Saitoh, N, Yoshiyama, K, Ihara, K, Seki, M, Ohtsubo, E, and Maki, H, 1999, Journal of Molecular Biology. Huang, L, Hu, E., Park, K, Lackovich, J, Lee, J., and Rashtchian, 2002, A. An ultra-specific PCR DNA polymerase. Manuscript in preparation. Sambrook, J, and D, Russell, 2001, Molecular Cloning: A Laboratory Manual Third Edition. Cold Spring Harbor Press, Cold Spring Harbor, NY. Smith, H, R, and S, M, Scotland, 1993, Isolation and identification methods for E. coli O157 and other Vero cytotoxin producing strains, Journal of Clinical Pathology, 46: 10-17. Smith, M.D, J, Jessee, T, Landers and J, Jordon, 1990, High Efficiency Bacterial Electroporation: 1x 1010 E. coli Transformants/mg. Focus 12: 38-41.2. Tarr, P, I, 1995, E. coli O157:H7: Clinical, diagnostic, and epidemiological aspects of human infection, clinical Infections, Dis. 20: 1-10. Read More