Cloning of the E. Coli Experiment - Essay Example

CLONING OF THE E. COLI Cloning of the E.coli is the experiment that is to be discussed in this report. The results of the experiment are as follows. The transformation efficiency of the E.coli was 4.104e2 Transformants / ng of DNA. The images for the experiments have also been shown though they are not as clear as expected. Tables have also been shown to represent the results either of the concentrations of the DNA of the plasmid and the genomic DNA concentration. Other results that have been tabulated are the transformation data for both the blue and white colonies. The writer obtained 171 total transformations in the control and zero transformations. This was used to calculate the transformation efficiency of the experiment.The aim of carrying out this experiment was to try out carry out cloning of E. coli and to calculate how efficient the cloning was. Key words The following are the key words that will be used in this report. To begin with is the word smid pALA2, which represents the plasmid to be used in the experiment. The other word is S. pombe. This in full is known as the Schizosaccharomycespombe. It is a species of yeast that is rod shaped and reproduces by fission. In this experiment on cloning, the yeast has been used in the determination of how the cell responds to DNA replication and destruction. The other key word in this report is cdc2, which stands for cell division control protein 2. This is also referred to by the name Cyclin-dependent kinase 1. It plays the role of encoding the fission yeast S.pombe in this experiment. List of tables & figures This section contains a brief description of the tables and figures that will be used in the report. They are as follows; figure one represents the gels showing the purified genomic DNA from S. pombe, compared to the provided DNA sample. Figure two is of the virtual gel showing restriction digest of purified plasmids. Figure three is of plasmid CPP, lane PCR product and purified PC. The forth figure is the control made of the Marker, KpnI cut pALA2 plasmid, the fifth is KpnI cut pALA2, which is a cdc2 orientation of the first plasmid. The sixth figure is the KpnI cut pALA2 that is also a cdc2 orientation of the second plasmid. The final figure is figure seven that shows the plasmid maps. Table one in the report shows the qubit values and the student number. Introduction Cloning can be defined in many ways though all these definitions will add up to the same thing. Cloning generally refers to the process of giving rise to organisms that are alike genetically either in nature like in the asexual reproduction of certain organisms such as bacteria, E.coli being amongst them.1 It could also be artificial by coming up with copies of DNA, cells or organisms. Cloning was first practiced in horticulture only, but currently the field has greatly advanced and more organisms can be cloned. 1. JanesPCR cloning protocols: importance of cloning Cloning has been of great importance since the time it was realized. Its uses include the following. One is the production of quality organisms. This has further led to the production of quality and quantity yields from these organisms. The organisms developed through cloning are disease resistant. This implies that these are of higher and more preferable quality.For instance, with cloning dairy cows can produce high milk quantity. Medicine is also using cloning in the treatment of certain diseases that could be difficult to treat. An example is the injection of brain cells of a fetus into the brain cells of a patient whose brain suffers from degenerative illnesses. Cloning is also of importance in the production of extinct and endangered organisms. For the extinct organisms, their DNA that was stored when it was still alive is used to produce a new organism. This has ensured that the organisms remain in the world without their extinction. In human beings, cloning has assisted couples that are infertile to get children. This is because cloning offers an ability to develop a whole organism with all the features that are in organisms that are sired in the normal manner. Cloning has also assisted in transplantation of organs. Body organs that are transplanted manage to develop in the receptor as a result of cloning. Gene cloning has been applied in forensics, whereby traces of DNA are used to produce more DNA that is used for further studies, such as detecting a criminal. Gene cloning has also assisted in genetic modification by placing a useful gene from an organism and placing it into another organism. Cloning can be carried out through various methods. These include the nuclear transfer method which involves the fusion of an adult animal cell with enucleated egg cell, so as form an embryo. This can be by microinjection which is a subdivision of fusion of the nucleus. The fusion of the nucleus to the cell is done by use of electric pulses. Artificial twinning is also a method of cloning that involves separation of an embryo into two or more embryos. The use of PCR In cloning is also another method used in cloning, whereby the PCR is used to amplify the DNA just as it is used to amplify RNA.2 Another method used is the restriction enzymes. This method makes use of enzymes in the cloning. 2. Janes and Chen PCR cloning protocols; PCR cloning Results After carrying out the experiment according to the procedure given, the following results were obtained. The results have been represented by tabulation of data, or theoretically. They are also presented in the order of the day when they were carried out. The following photograph was taken from the results obtained. The image was not clear for reasons that will be stated in the discussion of the findings. Figure one: This shows an image of the gels that contain the purified DNA. This was in a 0.8% agarose / 0.5 x TAE gel, using three lanes of the following order. 1:5 μL of DNA ladder, 2: genomic DNA sample 3: blank. The image above shows the purified DNA of the S. pombe, which is not very clear. This followed the isolation of the DNA from the S. pombe. The table below was filled to show the results for the experiment carried out on the second day. Table one: The table represents the concentration of plasmid DNA and the genomic DNA concentration. It shows the number of the student and the concentration in mg/L. This was after the use of a 0.8% agarose / 0.5 x TAE gel, with lanes containing gel in the following order lane 1:5 μL of DNA ladder lane 2:cut plasmid/treated plasmid sample lane 3:blank. From the table, it is evident that the highest concentration of the plasmid DNA is student number 2 with a concentration of 62.3 μL, while the lowest is at student 9 with 1.0 μL. The genomic DNA concentration ranges from student 2 to student 18 with 34.9 μL and 0.0 μL respectively. the average concentration of plasmid DNA was 25.61 with a standard deviation of 16.40. that of the genomic DNA was 7.61 and 11.26 in the same order. The student number 27 is the writer’s results. The image below was also taken from the experiment on day two. Figure two: This is an image of the purified DNA fragments that were observed after the utilization of a 0.8% agarose / 0.5 x TAE gel, with lanes containing gel in the following order lane 1:5 μL of DNA ladder lane 2:cut plasmid/treated plasmid sample lane 3:blank. The figure above shows the DNA fragments that were observed on this day. As can be seen, they are more visible and more detailed compared to the DNA that was purified on day one. The figure below was obtained after carrying out the experiment on the third day. Figure three: This is the image that was observed following the addition of 1% agarose / 0.5 x TAE gel and using three lanes of the gel loaded as follows; Lane 1:5 μL of DNA ladder (Hyperladder I), lane 2:tube CPP, lane 3:tube PCR, 4: Purified PC. The figure three is of the DNA after the PCR purification reaction. More details of the DNA are revealed compared to the other images observed above. For day four, the results are based on the transformation efficiency of the cells. These are calculated below. Using the following figures the calculation was done with the assistance from the website given. Concentration (in μg/μl) of the DNA used to transform: Ligation 0.00625 μg/Μl; Control 0.005 μg/μL, Volume (in μl) of DNA added to the transformation mix: Ligation 4μL ; Control 5μL. Volume (in μl) of the total transformation reaction: Ligation 300 μL; Control 300 μL. Volume (in μl) actually plated from the transformation mix: 5 μL. The number of colonies or transformants from the plate above: 171. After the calculation, the transformation efficiency was as follows. It was 4.104e5 Transformants / μg DNA or 4.104e2Transformants / ng of DNA. The table below shows the results for that fifth day’s experiment. Table two: Comprises of two tables all, showing the transformation data for the control and the ligation reaction. The tables are divided into the blue and white columns showing those that changed to these respective colors. From the figure, the highest transformation data obtained for the control and the ligation reaction are number 15 and 4 respectively for the blues while the whites were all equivalent at 0. The average for the total of the blues and white are 27,543 for the control reaction and 1,381 for the ligation reaction. The data the writer obtained was number 10, with 171 control reactions and 0 ligation reactions. Discussion It is considered easy to clone E.coli more than other organisms due to its unique characteristics. To begin with, it has the ability to hold other foreign DNA in it. Actually, it easily undergoes transduction and transformation with plasmids. It also has a very rapid growth rate when compared to other organisms, producing one generation in twenty minutes when provided with favorable growth conditions. Another reason is that it has unique methodologies of expressing proteins, making the expression of foreign proteins easy. For these major reasons, the experiment was majored on the cloning of this bacterium with plasmids. From the results obtained, the transformation efficiency is quite low, implying that there were quite a number of errors that were committed during the experiment. In the first day, there was the preparation of the preparation of LB agar plates. There was also the use of the Zymolyase enzyme and the Promega Wizard kit A1125 to prepare a genomic DNA extract, which would be used as a template for PCR reactions on the third day. The results obtained in the form of an image were not clear. It was expected that the image will be well visible but due to a mistake that was committed during the isolation of the genomic DNA from the S.pombe. Instead of carrying out all the expected centrifugations, the final one was skipped by mistake. The results of the second day were a clearer picture of the E.coli. The qubit values I obtained were low due to addition of the wrong amount of the reagents. In day two, the experiment comprised of the following. To begin with, the students were supposed to use the sigma Gen Elute PLN350 plasmid purification kit in the purification of plasmid pALA2 from E.coli that has been grown in the LB amphicillin liquid media prepared earlier on. There was also the qubit analysis of the plasmid and the genomic DNA concentration. This was aimed at estimating the concentration of DNA of the plasmid and the genomic DNA preparation using the qubitflourometer. Gel filtration was also carried out in order to remove the linear pALA2 DNA molecules, which would be used in the ligation reaction. In the third day, the following was done. Genomic DNA from S .pombe was used in the amplification of a specific S. pombe gene. PCR product purification was then carried out; whereby there was the utilization of the Sigma Gen Elute PCR clean up kit NA1020 so as to purify the DNA fragments from a PCR reaction mixture. The third day also comprised of an experiment that would cause restriction digestion of PCR fragments. Here, the NotI restriction enzyme was made use of to cut the purified PCR product ready for the cloning into plasmid pALA2. There was also the estimation of the Molar ratios of the PCR and cut pALA2 plasmid in preparation for a ligation reaction. In day four, there was first the preparation of LB ampicillin agar plates using X-Gal/IPTG solutions, which would be used in the selection of the blue or white E. coli. Ligation reaction is also carried out. Under this the gel purified pALA2 plasmid and digested PCR fragment were stuck together to form a new recombinant pALA2::cdc2 plasmid. There was also the transformation of the chemical competent E .coli cells. This was done using Bioline BIO-85036 Chemically Competent E. coli cells. A small amount of ligation reaction is added to the chemically competent E. coli cells, and transformation takes place by the cells taking up the intact plasmids thereby having the ability to grow on the agar selection plates. Another transformation is also carried out to obtain the transformation efficiency of the cells. This would be the control. The fifth day the potential recombinant plasmids are isolated. The E. coli colony that is suspected to contain the recombinant pALA2::cdc2 plasmids is identified. The plasmids from E. coli culture that could contain recombinant plasmids are also purified using the Sigma Gen Elute PFM10 plasmid purification kit. The isolated plasmids are also analyzed using the KpnI restriction enzyme, which cuts and maps potential pALA2::cdc2 recombinant plasmids. Generally, the experiment was not very efficient as was expected thus the transformation efficiency was low. To conclude, more care should be taken when carrying out this experiment so as to ensure that the results are good enough. This will also ensure that the images obtained are well visible in a way that one can draw conclusions from them. Though the experiment was a little hectic and tasky, there was the success in that there were some transformations that occurred on the bacteria. It is therefore recommended that there be more caution so as to avoid this low transformation efficiency. References Casali N. & Preston A. (2003) E. coli plasmid vectors: methods and applications. New York: Humana Press. Janes W. & Chen B. (2002) PCR cloning protocols. New York: Humana Press.Refworks. Theresa P. (2009). Top 5 Reasons E. coli is used for Gene Cloning.Biotech/biomedical, 27-35. Lipps G (editor). (2008). Plasmids: Current Research and Future Trends. Caister Academic Press Read More