Gene Cloning

Topics: DNA, Molecular biology, Restriction enzyme Pages: 6 (1802 words) Published: April 14, 2014
Cloning of plasmid pUC19 in E.coli bacteria

One aspect of the DNA cloning experiments that is carefully considered is the selection of cloning vectors. A variety of vectors have been created, each being suitable for a particular use. One common vector used in laboratories is a plasmid called pUC19. It is 2686 base pairs long and possesses an origin of replication which allows the production of over 100 copies in a competent E.coli cell. It possesses a multiple cloning site (MCS) which is artificially implanted by adding a polylinker sequence to it. The pUC19 plasmid is also altered by inserting a gene that codes for beta-lactamase which confers resistance to the antibiotic ampicillin (Read and Strachan 2011). The MCS occupies the 5’ end of the gene lacZ (Sherwood, Willey and Woolverton 2012). This gene codes for only the alpha-peptide of beta-galactosidase, an enzyme used to break down the disaccharide lactose into glucose and galactose (Read and Strachan 2011). The aim of this experiment is to incorporate a cDNA called CIH-1, from plasmid pBK-CMV, into pUC19. DNA cloning is dependent on type 2 restriction endonuclease enzymes. They function by cleaving both strands of DNA on specific points known as restriction sites. These sites are reliant on the sequences of DNA that are recognised by them. Different bacterial strains yield varying restriction endonucleases. There are currently over 250 recognition sequences identified (Read and Strachan 2011). Restriction endonucleases can cleave DNA sequences on vectors making them competent for the binding of other DNA fragments cut by the same enzyme. They are thus important tools in the production of recombinant DNA (Ahmed, Glencross and Wang 2011). The first objective of this experiment was to use two restriction endonucleases, EcoR1 and Xba1, to cut pUC19 and pBK-CMV. To ensure that the plasmids were successfully cut, analysis of the plasmid was carried out using gel electrophoresis. Gel electrophoresis is a method of separating DNA molecules using their sizes (Brown 2001). This is made possible due to the negative charge of nucleic acids. The DNA molecules are subjected to an electric field which makes them migrate toward the positive electrode (Hausman and Cooper 2013). The 2nd objective of this experiment was to construct recombinant DNA from pUC19 that was cut by EcoR1 and Xba1. The vector must undergo ligation in order to form the recombinant. This is achieved by using the enzyme DNA ligase, from the T4 bacteriophage, and ATP to form covalent phosphodiester bonds between annealed DNA molecules in the 3’ to 5’ direction. Ligation takes place at lower temperatures over a long period of time in order to allow optimal activity of DNA ligase (Holmes, Jones, Reed and Weyers 2007). The vector is then taken up by the host cells in a process called transformation. Transformation is an inefficient process as only a very small number of bacterial species can be easily transformed. As a result, the host cells have to undergo some form of physical and chemical treatment in order to make them competent (Brown 2001). E.coli was made competent by incubating it with MgCl2 to achieve the 3rd objective of introducing the recombinant pUC19 to them. Competent E.coli cells have altered cell walls which enable uptake of recombinant pUC19. Transformants can be identified using the selective marker. In the case of pUC19, this is the ampicillin resistance gene. For this reason, the transformed E.coli will be plated in agar containing the antibiotic ampicillin. In order to find transformants with recombinant pUC19, blue white colour selection was has been carried out. EcoR1 and Xba1 cut lacZ out of pUC19 to allow CIH-1 to ligate into it. For this reason, transformants without recombinant pUC19 cannot transcribe the alpha-peptide of beta-galactosidase resulting production of non-functional beta-galactosidase. Non-recombinant pUC19 has the 5’ end of lacZ intact and thus...
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