Biomedical Science Laboratory Report - Essay Example

? 216 biomedical science laboratory reports Sub-cloning of a foreign gene into the plasmid vector pUC19 and characterization of recombinant clones Introduction This practical utilized the plasmid vector pUC19, a common vector used for cloning small DNA fragments in Escherichia coli. pUC19 is 2686bp in total size and contain an origin of replication that enables the given vector to replicate in E.coli. pUC19 replicates in E.coli to produce approximately 100 copies per cell. In addition, the plasmid vector also contains an ampicillin resistance gene that encodes for B-lactamase, hence allowing E.coli to take up the transformants (plasmid) to be selected for growth on agar that got antibiotic ampicillin. It also got multiple cloning sites (MCS) in their plasmids which contain unique sites where several restriction enzymes like EcoR1 and Xba1 can bind and enable DNA fragments to be cloned there. MCS is often located within the gene lac Z’ that is responsible for encoding alpha peptidase belonging to the enzyme beta galactosidase. Usually, the remaining part comprising Beta galactosidase protein becomes encoded from a sequence found in the E.coli chromosome. It is the bacterial colonies that take up pUC19 which will express beta-galactosidase activity and take colour blue colour when grown in X-gal. On the other hand, when DNA fragments insert into MCS of pUC19, they create recombinant vector molecules which interrupts lac Z’ and hence the bacteria take up the recombinant plasmids and not produce beta-galactosidase resulting in production of white colonies. Restriction endonucleases enzymes are basically enzymes that cut DNA at specific sites. The enzymes cut at four, six or eight base pair sequences of which are palindromic. Sometimes restriction enzymes may cut in a middle of a recognition sequence, forming blunt ends, or commonly cut asymmetrically, resulting in sticky ends or stranded overhangs. DNA ligase come in hand to join the end pieces of the cut strands to create recombinant DNA molecule. Different restriction enzymes usually require different reaction medium for optimal activity. Buffer PH, salt concentration and magnesium ions are some of the common variables required by the enzymes to function. In order for one to assess the restriction digestion success, one needs to use a gel electrophoresis which can determine the generated sizes of the fragments. Since nucleic acids got negative charge, by the use of agarose gels in electrophoresis, they can be separated. DNA fragments usually move in relation to their length. Usually, small fragments migrate faster than large fragments. At the end, the formed DNA bands may be visualized from the gel by staining it with ethidium bromide and then observed using UV light. Aim of the experiment The aim of the series of the practical sessions was to transfer CIH-1, a fungal gene from pBK-CMV, a plasmid vector into pUC19, another plasmid vector. This was done by cutting the given fungal gene from pBK-CMV by use of Xba1 and EcoR1 that nick the plasmid from either side of the given fungal gene. The gene will then be ligated into pUC19. The ligated products eventually become transformed into E.coli and then there will be selection of bacteria colonies that will contain the recombinant pUC19 molecules. Confirmation Method In the first method involves restriction digestion of pBK-CMV and pUC19. In this method, one was provided with RDMM (a restriction digest master mix) that contains, water, a restriction digest buffer and the restriction enzymes XbaI and EcoRI. Ligation method is then followed. After it, agarose electrophoresis of the pBK-CMV and pUC19 was done. This was then followed by the preparation of chemically competent E.coli. XLI blue. Agar plates were then prepared Results The following were the results got after doing agarose electrophoresis of pBK-CMV and pUC19. From the electrophoresis sheet, one could realize that some base pairs shifted to a higher distance from the base line while others short distance. Therefore, the various distance between the top most part the base pairs reached from the base line recorded and values put in the table below. The results for the distance moved by the DNA size markers from the well included the following. DNA fragment size (base pairs) Distance moved from well (mm) 5000 10 3000 12 2000 15 1000 17 500 23 From the table above, it evident that DNA band with base pairs 5oo moved a distance of 23 mm, 1000 base pairs moved a distance of 17mm, 2000 base pairs moved a distance of 15mm, 3000 base pairs moved a distance of 12mm and 5000 base moved a distance of 10mm. From these findings, one can deduce that the shorter the base pairs, the longer the distance they travelled and vice versa. Discussion From the above experiment, it is evident that DNA fragments with lower base pair migrated longer distances than those with more base pairs. Restriction endonucleases enzymes are basically enzymes that cut DNA at specific sites. The enzymes cut at four, six or eight base pair sequences of which are palindromic. Sometimes restriction enzymes may cut in a middle of a recognition sequence, forming blunt ends, or commonly cut asymmetrically, resulting in sticky ends or stranded overhangs. DNA ligase come in hand to join the end pieces of the cut strands to create recombinant DNA molecule. Different restriction enzymes usually require different reaction medium for optimal activity. Buffer PH, salt concentration and magnesium ions are some of the common variables required by the enzymes to function. In order for one to assess the restriction digestion success, one needs to use a gel electrophoresis which can determine the generated sizes of the fragments. Since nucleic acids got negative charge, by the use of agarose gels in electrophoresis, they can be separated. DNA fragments usually move in relation to their length. Usually, small fragments migrate faster than large fragments. At the end, the formed DNA bands may be visualized from the gel by staining it with ethidium bromide and then observed using UV light. It is after that which one can be able to visualize the DNA bands on an electrophoresis sheet and measure the distance from the base line to where they have reached. Though such experiments are usually cumbersome to perform, one usually reaps a lot at the end by achieving sub-cloning of some part of DNA. The major aim of such experiments is to amplify ones DNA and be able to extract when in measurable quantity. Conclusion All in all, from the practical experiment, one could deduce that sub-cloning of a foreign gene into the plasmid vector pUC19 and characterization of recombinant clones is possible. Cloning of a given DNA is nowadays made possible by advancements in DNA technology. In this modern times, as medicine changes and a lot of DNA stuff is included as a diagnostic procedure for certain diseases, these techniques are proving to be more useful day in day out. It has come to our knowledge that genetics is a field that requires extensive research since it has shown much potential of improving many sectors. References Derrick, Almond, 2010, DNA engineering, New York: Insync publishers. Heaven, M and Donald,Switz, Microbial DNA cloning, New York: David and Blenk. Alex, Zurish, Restriction enzymes, Ney York: Macmillan. Peter, Zukab, Endonuclease, New York: Arnold, Kumar, Microbial practical work, London: Derrick publishers. Read More