Construction & Diagnostic of Recombinant DNA Plasmid - Research Paper Example

Molecular and Cell Biology Laboratory Mini Research Paper "Construction & Diagnostic of Recombinant DNA Plasmid" Abstract This paper is a presentation of the results of different laboratory experiments. One experiment led to the other. The target of laboratory one was to measure the level of competency exhibited by E. coli cells exposed to a chemical method of increasing competency. The chemical used was Calcium Chloride. The experiment established that the competency of cells increased greatly. In the next experiment, running PCR provided me with an amplified His3 gene which id used in subsequent steps to transform haploid yeast. Transformation occurred through homologous recombination. The experiment proved that specific integration does occur although cases of non-specific integration are rampant. In the next experiment, I constructed a sub-clone of the HIS3 gene and inserted it into a plasmid pSP72 making a recombinant plasmid which I used to transform bacterial cells. In order to determine whether the integration was successful, I extracted the plasmid to analyze if the inserted gene was present. However, I established that integration had not been successful. Introduction One of the laboratory techniques that help in understanding the basics of knocking out genes is the standardized procedure of replacing the ADE2 gene responsible for adenine biosynthesis with HIS3 that is responsible for one of the steps in histidine amino acids (Wach 1066). The procedure involves production of a hybrid polymerase chain reaction (PCR) as the first step. The hybrid product constitute of the vector DNA template and the primers of choice. Running of the PCR major steps produces the hybrid product. Agarose gel electrophoresis helps in determining whether PCR amplification occurred. The next step involves the transformation of yeast cells with the hybrid PCR product. The last step involves analysis of r4esults and morphology of transformed yeast cells. Other experiments involve transforming competent Escherichia coli cells with the use of a plasmid as a vector. This transformation follows the recombinant DNA technology protocol. The general procedure starts with digestion of plasmid DNA and template DNA of interest with restriction enzymes to generate DNA fragments with sticky ends. The second step involves ligation of the DNA fragments using DNA ligase, forming a recombinant plasmid. The next step involves insertion of the recombinant plasmid into the competent bacterial cells. The final step involves plating on appropriate media and selection of transformed cells. In addition, performing a backward procedure of isolating the plasmid from the transformed cells verifies insertion at the right locus. Laboratory 1: Transformation of Competent Bacteria Objective: Introduction of Plasmid DNA into E. coli cells and determination of transformation efficiency Materials and Reagents: Plasmid DNA Gene of interest SOC media LB amp media Procedure: The protocol preferred was the High Efficiency Transformation Protocol. However, a variation occurred with 2µl of plasmid DNA. The high efficiency transformation protocol requires thawing of competent cells in ice for about ten minutes. The next step involves transfer of 50µl of the cells to a transformation tube using a micropipette. Adding of 2µl of plasmid DNA into the tube followed. The next step involved placing the mixture on ice for 20 minutes. Next, exposure of cells to heat shock at 42ºC occurs, a process lasting 35 seconds. Following this was adding the right amount of SOC media to then cells. After this, incubation at 37Cº for 40 minutes and subsequent vigorous shaking followed. Plating of the cells in LB amp media and overnight incubation at 37ºC was the last step. In the first experiment, plating of the concentrated cell mixture without dilution occurred. In subsequent trials, there was dilution of cell solution at different dilution ratios. Results: Dilution factor Colonies counted Rounded to thousand Average 1:1000 680 680 1:10^4 35 350 1:10^5 7 700 1:10^6 0 0 Total 1730/3 576,000 Analysis of Results: Plating cells without dilution produced an extremely large number of colonies. Counting of these cells proved difficult and dilution offered better results. Too much dilution gives negative results because increased dilution reduced the probability of having transformed cells in the mixture. Transformation Efficiency calculations: Transformation efficiency refers to the number of units capable to form a colony that result from transformation of 1µg of plasmid into a specified amount of competent cells. However, in practical cases, transformation does not occur at such rates. In laboratory experiments, transformation of 100pg-1ng occurs. The super coiled plasmid undergoes transformation under ideal laboratory conditions. Transformation efficiency is useful in making ligation comparisons. Formula for calculation is: Transformation efficiency = colonies/µg/dilution. Transformation efficiency (TE) TE= Total # of bacteria growth in the plate Total amt. of plasmid DNA spread on plate Ci = 2 μl plasmid DNA used and the stock concentration was [10pg/ μl] Total added in the original tube 20pg Vf= 1mL Starting concentration 20pg/ml Diluted to 1:1000 (10^3) Plated (spread) 100μl = 0.1ml Final concentration 20x10^-3pg/ml (20x10^-3pg/ml) / (0.1ml) = 2x10^-3pg Amount of DNA plated = Cf = 2x10^-3pg TE = 576 x 10^3 transformants/ 2 x10^-3pg (10^6pg/ug) = TE = 2.88 x 10^14ug Discussion of Results: After dilution, it was possible to count colonies and then calculate the transformation efficiency as shown above. The transformation efficiency implies success of the experiment because colonies observed indicate presence of transformed cells. Only cells containing the recombinant DNA plasmids survive on the LB amp media. Growth of these cells is possible because cells obtain an ampicillin resistant gene. Laboratory 2: Polymerase Chain Reaction Amplification Section 1: hybrid PCR product preparation using two 60-mer primers- amplification and electrophoresis Objectives: 1) Preparation of hybrid PCR product using 60-mer primers and DNA templates through PCR amplification-amplification of HIS3 gene flanking it with sequences to ensure target integration 2) Running agarose gel electrophoresis to visualize PCR products Materials and Reagents 60-mer primers Plasmid DNA template Buffer Master Mix Magnesium Chloride Blue Loading Dye Taq polymerase Procedure: The procedure involved preparing a master mix sufficient for six samples. There were no primers in the master mix. The buffer required for this procedure was the ten times standard polymerase buffer with a maximum final concentration of 1 x (1.5 mM MgCl2). However, in order to establish the effect of magnesium chloride, an alteration to its concentration occurred for sample 4. In this sample, the amount of MgCl2 added was 4µl of 2mM Mg++. The template DNA amounted to s20ng/µl of the plasmid pF6a-HIS3. The procedure starts with pipeting 40µl of the Master Mix into PCR tubes containing the primers. Running PCR involved four major steps. The first step was denaturation of the DNA leaving it single stranded. This occurred in one cycle at the temperature range of 94-95ºC. The second step anneals the primers to the ends of the single stranded DNA and took place in 30 cycles, for five minutes. The temperature ranges included, 94ºC for 30 seconds, 49ºC for 45 seconds and 72ºC for 1.5 minutes. The third step in PCR is the extension of DNA and occurred at 72ºC for seven minutes. The last step took place at 4ºC and served to ensure a strong hold of the reaction. After PCR, running an agarose gel electrophoresis followed with 10µl from each sample loaded onto the gel. Adding of blue loading dye to the 10 µl of each sample is critical before loading to the gel. Included in the gel was a molecular ladder. Distribution of samples loaded onto the gel: Samples # Description 1 Both primers+40μl master mix 2 Both primers + 40μl master mix 3 Both primers + 40μl master mix 4 Both primers + individual vol. Reaction. + 4μl of 2mM Mg++ (results showed in gel) 5 5μl R1 + 5μl H2O + 40μl master mix 6 5μl F1 + 5μl H2O + 40μl master mix 7 Nothing 8 Molecular weight (ladder) Results of the experiment: Figure no. 1: gel showed the results for the band of sample number 4. A close examination of the gel results portrayed bands in sample four only. In gel electrophoresis, products separate according to size and charge. Negatively charged fragments migrate to the positive side of the gel and vice versa. DNA fragments have negative charge and migrate to the positive side. The fragment in sample four moved 3.8cm along the gel. The fragment was 1.5 kilobase, 1,517 base pair and had DNA mass of 57ng. There are varied explanations elaborating why the other samples formed no bands. It is possible that human error occurred but the most valid reason lies in the effect of Magnesium Chloride concentration. In addition, one can infer that the master mix was deficient of some essential component. Table #3: Data of the DNA ladder fragments/Kb/bp/DNA mass Fragment Dist. cm Kb Bp DNA mass (ng) 1 1.8 10 10,002 40 2 2.0 8 8,001 40 3 2.2 6 6,001 48 4 2.35 5 5,001 40 5 2.5 4 4,001 32 6 2.9 3 3,001 120 7 3.4 2 2,017 40 8 3.8 1.5 1,517 57 9 4.1 1.2 1,200 45 10 4.45 1 1,000 122 11 4.6 0.9 900 34 Graph #1: Fragments size Kb vs. Distance traveled (cm) Discussion: PCR was successful in sample four only because visualization of the PCR products via gel electrophoresis showed that a fragment of 1517 base pairs formed band 3.8 cm distance on the gel. The other samples prepare using the master mix produced no results and this implies that the mix did not meet the minimum PCR requirements. Other groups trying out different MgCl2 concentrations and band formation did not occur. The concentration of magnesium chloride is a critical aspect in the polymerase reaction. Laboratory 3: PCR Clean up, Yeast Transformation with Lithium Acetate Section 2: Transform Yeast with Hybrid PCR Product Objective: 1) cleaning up PCR product from sample four with Exosap-IT kit 2) Cell transformation: 1) with no DNA 2) 20 µl of PCR product. 3) 50 µl of PCR product Materials and reagents: Lithium acetate Sterile water Micro centrifuge tubes Micropipettes PEG Salmon sperm DNA Procedure: The first step involved culturing of Saccharomyces cerevisiae HIS3 mutant cells (haploid) and harvesting of the cells at 1-2 x 107 cells/mL (O.D.600nm of 1:10 dilution = 0.1-0.2. The next step entailed harvesting 25mL of yeast culture by centrifugation at 3000xg in a sterile tube. Centrifugation lasted 5 minutes. Decantation of the medium and resuspension of the cells in 25 Ml of sterile water then occurred. Pelleting of 7yeast cells at 3000xg for 5 minutes followed. In the next step, decantation of water together with cell resuspension in 1mL of 100nM Lithium acetate occurred and transferred to a sterilized 1.5mL microcentrifuge tube. Pelleting of cells at top speed for five seconds then followed. Using a sterilized micropipette to remove the lithium acetate was the next step. Subsequently, resuspension of cells to a final volume of 250µl and addition of about 200µl of 100Mm LiAcetate formed the final suspension at 2 x 109 cells/mL. The next step involved vortexing and pipetting of 50µL aliquots putting them into labeled and sterilized micro centrifuge tubes. A repeat of pelleting and removal of acetate followed. In the next step, addition of the following reagents occurred: • 240μL PEG (50% w/v) • 36μL 1.0M lithium acetate • 25μL SS-DNA (salmon sperm “carrier” DNA 2mg/mL) • 50μL total volume PCR product + water (or only water for no DNA control) Vigorous vortexing for each tuber followed to allow complete cell resuspension. Incubation of the tubes at 30ºC for 20-30 minutes was the next step. Exposure of the cells to heat shock at 42ºC for 20 minutes followed and subsequent centrifugation at 6-8000rpm and removal of transformation mix with using a sterilized micropipette. Addition of 0.5mL of sterile water and gentle resuspension of pellet by up and down pipeting formed the step. Subsequently, plating followed and involved spreading of 200µL of each transformation onto plates and storing in refrigerator. After absorption of liquid, inversion of plates and incubation at 30ºC for 2-3 days followed in the last step of the experiment. Distribution on plates for transformation: Plate 1: 50ul cells + no DNA + 50ul ddH2O Plate 2: 50ul cells + 20ul DNA from our pcr product + 30ul ddH2O Plate 3: 50ul cells + 50ul DNA pcr product from professor Calculations: Starting concentration yeast cells (1.61x10^7cells/ml) x (25ml) = 4x10^8 total cells/0.25ml in the tube= 1.6x10^9cells/ml Results observations: Yeast analysis Figure #2: shows result of the transformation; Plate 2: 50ul cells + 20ul DNA from the group’s PCR product + 30ul ddH2O Counted colonies: Plates # Red colonies White colonies 1 None None 2 7 11 3 3 7 Total 10 18 Whole class: Group # Red colonies White colonies 1 9 18 2 3 8 3 11 13 4 7 19 5 our 10 18 Total 40 76 Transformation result score (TRS): # of red colonies/#of total colonies: For group #5: TRS: 10/28= 0.36 transformants For the whole class: TRS: 40/76 = 0.53 transformants. As for the whole class: TRS: 40/76= 0.53 transformants The number of pink colonies was relatively small and this is because of inappropriate adenine concentration. Since deletion of ADE2 gene occurred, the cells needed adenine –in order to grow. Insufficient adenine results to a small number of pink cells. The pink color is because of p-ribosylamino imidazole accumulation since lack of ade2 gene cuts short adenine biosynthesis pathway. On the other hand, red colonies indicated that gene knockout took place and that the cells had enough adenine for growth. White colonies implied non-specific integration of the his3 gene. Non-specific integration placed he gene on a different locus and not the place of ade2 gene. Discussion: The white colonies appeared in large numbers indicating that non-specific integration took place in most of the cases. The first plate that did not lacked colonies and served as a good control. Despite the fact that red colonies were relatively small, it was a positive implication of specific gene knockout integration of his3 gene at ade2 locus. Pink colonies proved that gene knockout occurred. The cells lacked the ade2 gene and needed adenine to grow. Insufficient adenine cuts short the adenine biosynthesis chain. The growth of white colonies presents gene therapy. Biosynthesis of purine nucleotides and steps at which different yeast genes function (www.phys.ksu.edu/gene/GENEFAQ.html): Laboratory number 4: Subclone PCR Fragment, Bacterial Transformation Objective: construction of recombinant plasmid and insertion into competent bacterial cells Materials and reagents: ExoSAP-IT kit reagent Plasmid vector pSP72 Enzymes EcoRI and Xma1 Procedure: The first step involved cleaning up PCR product number 4 as described in laboratory three. The other steps involved digestion of plasmid and PCR product using restriction enzymes EcoRI and Xma1. After digestion with these enzymes, the next step involved cloning of the PCR product into the vector. Before the cloning step, inactivation of the enzymes at 65C for 20 minutes occurred. After heat inactivation, running a gel electrophoresis confirms whether the enzymes cleaved the PCR product and the plasmid DNA. Cleaving and electrophoresis should run at the same time. Confirmation of DNA cleavage is by observing the changing plasmid DNA from a circular shape to linear fragments. The PCR product forms bands of smaller fragments on the gel. Ligation of the fragments using DNA ligase is possible because digestion with similar enzymes produces sticky ends. Ligation serves to bind the fragments of the circular DNA with those of the PCR product. For digestion of the vector DNA, the group needed 1.5μl Vector DNA, 1.5µl buffer #4, and 1.5µl of BSA, 0.75μl EcoR1, 0.75μl Xma1, 9μl ddH20, and 15μl final volume. For the PCR product, the group utilized 10μl PCR fragment DNA, 1.5µl buffer, 1.5µl of BSA, 0.75μl EcoR1, 0.75μl Xma1, and 14.5μl final volume. In both cases, incubation of the mixtures at 37C for about an hour followed. Heat inactivation to stop enzyme activity formed the last step in digestion (Mulhardt 40). Addition of the T4 ligase enzyme followed and then incubation for ten minutes. DNA ligation protocol: Materials and reagents: Tris T4 DNA ligase DTT Polyethylene glycol ATP Magnesium Chloride Acetylated BSA Oligonucleotide Procedure: The first step involves treatment of vector fragments with alkaline phosphatase to in order to remove 5’phosphates. This serves to block self-ligation. Step two involved addition of the control that lacked DNA. Addition of the DNA ligation followed. Addition of PEG reduces the level of transformation (Bartlett and Starling 137). Molar Ratio Calculation: In this experiment, the molar ratio was 1: 6 depending on the calculation of the vector and PCR product in use. From the gel electrophoresis bands, determination of distance moved by PCR product, vector, and molecular ladder was done. PCR fragment was 100ng, which is 1.5µ L of PCR fragment while the vector was 50ng. From this inference, the ratios needed adjustment to1: 6. Calculation: 1:1 ratio 50ng/2400bp vector: x/1400bp insert = 30ng The insert is three times l-better than the PCR product. Therefore then ratio for use becomes 1:6 Vector: 1.5µg = 50ng, Insert: 3.0µg = 90ng COMPONENTS: Component 20μL Reaction Your calculated volume 10X T4 DNA Ligase Buffer 2 μL 2 μL Vector DNA (50 ng) 2.4kb 0.03 pmol 1.5μL Insert DNA (10X molar excess over vector)1.4kb 0.1 pmol 3.0 μL ddH2O to 20 μL 12.5 μL T4 DNA Ligase 1 μL 1μL Transformation protocol into CaCl2 competent cells: 50µL of C2988 bacteria was subject to transformation in a transformation tube. Addition of 10µL of sample from reaction followed. Icing of the mixture and exposure to heat shock for 30 seconds was the subsequent step. Repeated icing for 3 minutes followed. In the next step, the group added 950 µL of LB media into the mixture and incubation at 37ºC for 10 minutes. Plates: Plate #1: 200µl cells + insert Plate #2: 100µl cells – insert Plate #3: 50µlcells + insert Results after observed the plates: Plate #1: 200µl cells + insert = Uptake the plasmid Plate #2: 100µl cells – insert = no colonies and was a good control Plate #3: 50µl cells + insert = Bacteria uptake plasmid Growing of 200µL from plate number one followed in preparation for the experiment on mini prep extraction. Discussion: Bacteria can naturally take up naked DNA if polymerases in the bacteria have the capacity to recognize the origin of the DNA. In other cases, exposure to some chemicals such as calcium chloride makes the cells swell, a state which increases chances of incorporating the gene of interest into the cells. The advantage of induced transformation is the fact that scientists can select the gene of interest. In this case, addition of a vector that carries the gene of interest into the competent cells into a transformation tube forms the first step. A very crucial step follows which involves exposing the mixture to heat shock at 42ºC. At this temperature, the cells make use of hest shock genes to survive and the intake of the DNA is at its optimum. Higher temperatures would kill bacteria. Laboratory 5: Plasmid DNA, Restriction Digest Objective: Extractions of the recombinant plasmid DNA from the transformed bacterial cells and then digests with restriction enzymes to verify the presence of the inserted his3 gene Background information: The procedure require the separation of chromosomal and plasmid DNA through alkaline treatment which leads to the breakage of Hydrogen bonds. Alkaline p.H ranges lead to denaturation of DNA and RNA degradation. However, plasmid DNA is resistant to denaturation by alkaline conditions and returns to its original state when p.H reduces. The circular DNA shape does not change and chromosomal aggregates. Elusion of the plasmid depends on charge because the negatively charged plasmid DNA moves to the positively charged membrane filter. Column purification follows. Diagnostic Restriction Digest Protocol and Information: DNA isolation using the ultra clean mini-prep plasmid kit produced the recombinant DNA. In order to establish information essential for use in determining the restriction enzymes, the first step was mapping the sequence on a restriction-mapping program. The folder mapped sites on the DNA. BAMh1, Xba1, and Pst1were the available enzymes for the restriction digest. However, Pst1 proved the most appropriate and cleave the sequence at 31bp, 286bp, and 733bp. The sequence was 3760bp. The website neb.com provided information concerning the right buffer for used and this was the buffer number 3. Preparation of DNA sample and vector sample followed as per the protocol and adjusted to a final volume of 20µl. the control of the experiment was an uncut vector and vector digest. In the next step, loading of the sample, the control and a molecular weight ladder onto a gel to visualize the bands formed. The preferred gel is 1% agarose. Results observed from the gel: The samples were then loaded in the agarose gel in the following order: Legend: MW= ladder, 1= samples from a laboratory partner. 2 = own samples, V= vector, M = miniprep 1: MW-2: Uncut vector-3: V digest1- 4: V digest2- 5: M.uncut1- 6: M.digest1- 7: M.uncut2- 8: M.digest2 Figure #3: Agarose Gel diagnostic digests results. The results for showed in line 7 and 8 were better seem under the UV light. Discussion Gel electrophoresis result analysis portrays formation very faint bands. The expected resulted included two bands because Pst1 cleaves the fragment once at 866bp. According to the results of the working sample, the results were negative. However, there probably was a cause of error because the sample used did not show sufficient pellets during mini-prep DNA isolation. A repeat of the same experiment with a different sample would probably produce positive results. Conclusion The transformation of bacterial cells requires a pre-exposure to Calcium Chloride in order to increase the chances of DNA uptake. Gene knock out is a possibility and the yeast cell proved that specific gene knock out and therapy can occur. The recombinant DNA technology has a revolution in molecular biology. Gene therapy can be a viable solution to genetic disorders if research addresses the issue of ensuring maximum specificity. Most of the experiments realized their objectives. Work Cited Mulhardt, Cornel. Molecular biology and genomics. Massachusetts: Academic Press, 2007. Print. Ratledge, Colin. Basic Biotechlogy. Cambridge: Cambridge University Press, 2006. Print. Stirling, David and Bartlett, John. Pcr protocols. New York: Human Press, 2003. Print. Wach, Achim et al. Heteroloogous HIS3 Marker and GFP Reporter Modules for PCR- Targeting in Saccharomyces cerevisiae. Read More