Cloning the CDC2 Gene from Schizosaccharomyces Pombe - Term Paper Example

Cloning the cdc2 gene from Schizosaccharomyces pombe into Escherichia coli. By (Insert both names) (Name of class) (Professor’s name) (Institution) (City, State) (Date) Abstract Cloning is a molecular technique of making identical copies of a gene. Restrictive enzymes, for example, the Not1 are used to cut genes. A polymerase chain reaction (PCR) can then be used to amplify desired gene. High fidelity PCR is used when one wants to get low errors in their results. High fidelity restriction enzymes are enzymes which cut the DNA and the vector at specific points with accuracy. Gel electrophoresis is used to observe different segments of restricted DNA fragments based on their size. A screening technique, that can be used to detect the gene in the smid pALA2, is called blue-white screen. The media contain x-gal which is colourless, an analog of lactose that forms a blue color. This paper demonstrates the efficiency of high fidelity PCR in the cloning of cdc2 ORF gene from S. pombe into E. coli. The cloning of CDC2 genes of S. pombe was successfully inserted into E. coli. It is used to bring an improved knowledge of the galactose gene in S. pombe. The results show that gene was successfully cloned. It could be visible in the gel through electrophoresis bands. The blue color in the media also indicated the presence of beta galactosidase. Keywords: smid pALA2, cdc2, S.pombe. List of abbreviations: DNA: Deoxyribonucleic acid. PCR: Polymerase Chain Reaction. ddH2O: double-distilled water. S.pombe: Schizosaccharomyces pombe. E.coli: Escherichia coli. X-Gal: 5’ Bromo-4-chloro-3-indolyl-b -D-galactopyranoside. IPTG: Isopropyl-beta-D-thiogalactopyranoside. LB agar: Luria Broth agar.. Plasmid pALA2: it contains the kanamycin resistance gene in the EcoR1 site of plasmid pUI1015 CDC2 - cell division cycle (cdc) genes in yeast Schizosaccharomyces pombe that appears to be a central positive regulator. Not1 (restriction enzyme): it is an invitrogen restriction enzymes List of tables & figures: Figure 1: gels show the purified Genomic DNA from S.pombe. Run compared with provided DNA sample. Table 1: Excel graph of class DNA concentrations showing Qubit values against student number. Highlight which are your data and Class Plasmid purification data and your data. Figure 2: virtual gel showing Restriction digest of purified plasmids Figure 3: plasmid CPP, lane PCR product and Purified PC Figure 4: Marker, KpnI cut pALA2 plasmid Figure 5: KpnI cut pALA2: cdc2 orientation 1 plasmid. Figure 6: KpnI cut pALA2 : cdc2 orientation 2 plasmid. Figure 7: Plasmid maps Introduction Cloning is a molecular technique of making identical copies of an organism or part of the organism. It involves properly selecting the desired gene for cloning. A gene is a section of a DNA that is responsible for producing a specific protein (Hoffman, 1990). These genes are then cut using restrictive enzymes. There are different types of restriction enzymes, for example, the Not1 restriction enzyme. When isolating DNA it is essential to separate DNA from RNA. This is made possible by using RNase which digests RNA. A polymerase chain reaction (PCR) can then be used to amplify desired gene (Nelson, 2005). This gene is then inserted into a vector which has specific restriction sites using both restriction enzymes and ligation enzymes. High fidelity PCR is used when one wants to get low errors in their results. The sequence of such genes needs to be very accurate. Proof reading enzymes, which have 3’ to 5’ exonuclease activity, are used. High fidelity restriction enzymes are enzymes which cut the DNA and the vector at specific points with accuracy (Nelson, 2005). Where blunt edges are produced, primers can be added which insert restriction sites to the gene. Ligation enzymes are used to join two pieces of DNA (Hoffman, 1990). The vector then acts as a vessel to move the desired gene into another organism. The organism can then be screened for the presence of the desired gene. Gel electrophoresis is used to observe different segments of restricted DNA fragments based on their size. A screening technique, that can be used to detect successful ligation of the gene to the vector, is called blue white screen. The vectors are then transformed into E. coli and then grown in a media. The media contain x-gal which is colourless is an analog of lactose that is cut by beta galactosidase to produce 5 bromo 4 chloroindoxyl which when formed is blue in color. This confirms the presence of active beta galactosidase. Colonies, which are blue, indicate vectors with lac z alpha which is interrupted (Nelson, 2005). White colonies indicate the presence of lac z alpha. The lac z alpha disrupts the formation of active beta galactosidase. Plasmid vectors used in the blue/white screening possess a multicloning site that is carefully inserted in the regions that code the lac z gene is located. Cell division cycle (cdc) genes that fuse Schizosaccharomyces pombe are the cdc2. These genes have been shown to have a positive central regulatory function. At certain temperatures, cdc2 arrests the cell division at G1 and G2/M points. A protein kinase called p34 is the product formed by cdc2 genes. pALA2 is a plasmid which has a gene which is kanamycin resistant in the ECOR1 site. Such plasmid can be observed when they are made to grow in a media that contains kanamycin antibiotic (Hoffman, 1990). This paper will demonstrate the efficiency of high fidelity PCR in the cloning of cdc2 ORF gene from S. pombe into E. coli. Method and Materials As per the laboratory manual. Results The specificity of cdc2 PCR amplification and efficiency of purification of the PCR product was determined via agarose gel electrophoresis. An intense band of approximately 23.113kb was detected in the PCR reaction as is shown in figure 1 below. The molecular weight of this band corresponded to the expected molecular weight of the cdc2 ORF amplified using the forward primer SPBC11B10.09-F and reverse primer SPBC11B10.09-R. The intensity of the band indicated that the cdc2 gene had been efficiently amplified. After purification, the intensity of the cdc2 PCR product was visible using the gel electrophoresis. This indicates that the cdc2 had been successfully cloned. The final concentration was approximately 20ng/μL. The band may not be clear due to the thickness of the gel. It might have been too thick preventing proper movement of the DNA. The purity of the DNA might also have been compromised. Figure 1: Gels showing the purified Genomic DNA from S.pombe. run compared with a provided DNA sample (Molecular weight = 23130 bp) The plasmid and genomic DNA concentration of the purified plasmid E. coli was analyzed using the Qubit flourometer. Table 1 below indicates the various concentrations. The average plasmid DNA concentration was 25.61mg/L while the standard deviation was 16.4. The average genomic DNA concentration was 7.61mg/L while the standard deviation from the group was 11.26. The results show that the plasmid DNA concentration was more that the genomic DNA concentration. Table1: Excel graph of class DNA concentrations showing Qubit values against student number Plasmid DNA concentration Genomic DNA concentration Student number Mg/L Student number Mg/L 1 33.7 1 20.1 2 62.3 2 34.9 3 25.6 3 25.3 4 2.7 4 0.0 5 35.6 5 0.4 6 28.7 6 2.4 7 38.4 7 4.0 8 4.1 8 2.0 9 1.0 9 30.0 10 1.3 10 0.0 11 22.2 11 0.0 12 4.9 12 5.9 13 20.1 13 0.1 14 12.8 14 0.0 15 53.9 15 2.3 16 28.6 16 0.7 17 14.8 17 0.7 18 31.7 18 0.0 19 37.5 19 0.7 20 16.5 20 1.6 21 37.9 21 6.0 22 45.3 22 3.4 23 43.1 23 4.2 24 12.6 24 0.7 25 22.3 25 25.0 26 28.2 26 27.4 27 27 Average 25.61 Average 7.61 Std deviation 16.4 Std deviation 11.26 The efficiency of restriction enzymes (Not1) using PCR amplification of restricted and efficiency of purification of the PCR product was determined via agarose gel electrophoresis. An intense band of approximately 10.003kb was detected in the PCR reaction as is shown in figure 2 below. Gel purification was used to remove linear pALA2 DNA. Alkaline phosphate treatment was used to prevent re-ligation. Figure 2: virtual gel showing Restriction digest of purified plasmids Molecular weight = 10037 bp The S. pombe genomic DNA and PCR was used to amplify a purified specific s. pombe gene. Figure 3 below shows the results. The molecular weight of each band was 1.302kb. Not1 restriction enzyme was used. The plasmid CPP, lane PCR and the purified PC indicated the same molecular weight. Figure 3: plasmid CPP, lane PCR product and Purified PC Molecular weight = in both lane 1302bp In the screening technique using the blue/white selection, table 2 shows the results. In control, the total number of blues colonies was 743655 while the average was 275343. There were no while colon in the control. In the ligated reaction, the total number of blue colonies was 37277 while the average was 1381. Table 2: table showing the observed blue and white colonies Control reaction Class transformation data Ligation reaction Class transformation data Efficiency of insert cloning Student number Blue White Total Student number Blue White Total 1 33240 0 33240 1 406 0 0 5.3e2 2 10440 0 10440 2 214 0 0 4.4e2 3 1600 0 1600 3 20 0 0 1.3e2 4 42480 0 42480 4 18000 0 0 4.4e2 5 23040 0 23040 5 7 0 0 2.3e4 6 6160 0 6160 6 29 0 0 14.3e1 7 55200 0 55200 7 2775 0 0 42.3e2 8 1620 0 1620 8 45 0 0 4.33e2 9 33000 0 33000 9 338 0 0 4.34e2 10 171 0 171 10 0 0 0 4.3e52 11 105 0 105 11 94 0 0 4.3e26 12 70800 0 70800 12 4350 0 0 64.3e2 13 3636 0 3636 13 383 0 0 44.3e2 14 48720 0 48720 14 814 0 0 84.3e2 15 212400 0 212400 15 9 0 0 24.3e2 16 6300 0 6300 16 300 0 0 54.3e2 17 1293 0 1293 17 178 0 0 24.3e2 18 6420 0 6420 18 225 0 0 14.3e2 19 23880 0 23880 19 6 0 0 34.3e2 20 51840 0 51840 20 600 0 0 44.3e2 21 38040 0 38040 21 28 0 0 44.3e2 22 2688 0 2688 22 22 0 0 54.3e2 23 516 0 516 23 233 0 0 14.3e2 24 11040 0 11040 24 129 0 0 64.3e2 25 25200 0 25200 25 270 0 0 64.3e2 26 6 0 6 26 2 0 0 14.3e2 27 3360 0 3360 27 7800 0 0 34.3e2 Total 743655 0 743655 Total 37277 0 0 Average 27543 0 27543 Average 1381 0 0 Std Deviation Std Deviation The transformation efficiency was 4.3e2 Transformants / μg DNA. Discussion The specificity of cdc2 PCR amplification and efficiency of purification of the PCR product was determined via agarose gel electrophoresis. An intense band of approximately 23.113kb was detected in the PCR reaction as is shown in figure 1 below. The molecular weight of this band corresponded to the expected molecular weight of the cdc2 ORF amplified using the forward primer SPBC11B10.09-F and reverse primer SPBC11B10.09-R. The intensity of the band indicated that the cdc2 gene had been efficiently amplified. After purification, the intensity of the cdc2 PCR product was visible using the gel electrophoresis. This indicates that the cdc2 had been successfully cloned. The final concentration was approximately 20ng/μL. The band may not be clear due to the thickness of the gel. It might have been too thick preventing proper movement of the DNA. The purity of the DNA might also have been compromised (Hoffman, 1990). The plasmid and genomic DNA concentration of the purified plasmid E. coli was analyzed using the Qubit flourometer. Table 1 below indicates the various concentrations. The average plasmid DNA concentration was 25.61mg/L while the standard deviation was 16.4. The average genomic DNA concentration was 7.61mg/L while the standard deviation from the group was 11.26. The results show that the plasmid DNA concentration was more that the genomic DNA concentration (Lubert, 2002). The efficiency of restriction enzymes (Not1) using PCR amplification of restricted and efficiency of purification of the PCR product was determined via agarose gel electrophoresis. An intense band of approximately 10.003kb was detected in the PCR reaction as is shown in figure 2 below. Gel purification was used to remove linear pALA2 DNA (Lubert, 2002). The size of plasmid = 8220bp and the gene size=1189bp would be shown in the bands as distinct as possible compared to the others (Lubert, 2002). The orientation of a gene can be determined when several restriction enzymes are used. Two or more restriction enzyme means that more than one place will be cut. The possibility of joining back is also prevented if restriction enzymes are used since the sticky ends will not be compatible. For efficient ligation to take place, the ratio of plasmids and DNA fragments must be right. The enzyme ligase must also be included in the reaction for effective ligation. Therefore, if the concentration of the DNA fragments is not reached, then the number of bands observed during electrophoresis will not be distinct (Lubert, 2002). A screening technique that can be used to detect successful ligation of the gene to the vector is called blue white screen. The vectors are then transformed into E. coli and then grown in a media. The media contain x-gal which is colourless is a resemblance of lactose that is cut by beta galactosidase to produce 5 bromo 4 chloroindoxyl which when formed is blue in color. This confirms the presence of active beta galactosidase (Lubert, 2002). Colonies which are blue indicate vectors with lac z alpha which is interrupted. White colonies indicate the presence of lac z alpha. The lac z alpha disrupts the formation of active beta galactosidase. Plasmid vectors that are to be used in the blue/white screening possess a multicloning site that is carefully inserted in region that codes for lac z gene. pALA2 is a plasmid which has a gene which is kanamycin resistant in the ECOR1 site. Such plasmid can be observed by growing them in kanamycin antibiotic containing media. The presence of the antibiotic converts the colonies into white color. The multi colon site contains several antibiotics which can be detected using several methods (Lubert, 2002). Isopropyl thiogalactoside or isopropyl beta D thiogalactopyranoside (IPTG) is used together with xgal in the selection of colonies. It is effective used when detecting bacteria such as E. coli. It is a stable synthetic lactose analogue which makes the lac repressor inactive. It also makes the beta galactosidase is synthesized (Lubert, 2002). The class results indicate that it was more efficient to extract the plasmid DNA than the genomic DNA (Lubert, 2002). Errors that could have occurred in the experimentation could have occurred during pipeting of the various solutions either into the epidoffs or into the gel. These errors led to change in concentrations of the DNA. The concentration changes also affect the various reagents that have been used (Lubert, 2002). The cloning of CDC genes of S. pombe was successfully inserted into E. coli. It is used to give understanding of the cdc2 gene in S. pombe (Lubert, 2002). References Hoffmann, A. (1990). Cloning of the S. pombe TATA gene. Cold Spring Laboratory Press Lubert, S. (2002). Biochemistry. The 5th edition. W. H. Freeman Company. Nelson, D. L. (2005). Principles of Biochemistry, (4th edition), W. H. 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