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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 transformants with that plasmid produce functional beta-galactosidase.
This can be detected by adding 5-bromo-4 chloro-3-endolyl –beta-D-galactopyrosinoside (X-gal) into the agar plates. X-gal is an analog of lactose which is broken down by beta-galactosidase to produce a blue-coloured product (Sherwood, Willey and Woolverton 2012). For this reason, the transformants possessing non-recombinant pUC19 will produce blue colonies whereas transformants, with recombinant pUC19 will produce white colonies. Isopropylthiogalactoside (IPTG) was also added to the agar in order to induce the transcription of beta-galactosidase. IPTG works by binding to the repressor protein inactivating it (Read and Strachan 2011). Results
In figure 1, with the pBK-CMV plasmid, there are two DNA fragments shown as bands on the electrophoresis gel, one band which suggests a fragment size of approximately 5000 base pairs and one with 500-100 base pairs. These are within range of the predicted band sizes for pBK-CMV. The data collected from the gel electrophoresis gel regarding Puc19 produced only 1 band with the fragment size of roughly 3000 base pairs. This is close to the predicted size of the Puc19 if it has incorporated the CIH-1 molecule (2664+600 = 3264). Table 1 shows the number of colonies of the transformation plates of 3 different samples. Tube 2 which is the positive control, tube 3 which is the negative control and tube 1 which is the colony subjected to transformation and ligation. Dilutions of competent cell colonies are also shown. Tube 1 possessed more white colonies than blue colonies which suggest that most of the competent cells have undergone successful transformation.
The colonies produced from tube 2 and 3 are only white as there were no transformation of Puc19 as predicted. Figure 2 shows the results of the separation of DNA fragments from the plasmid DNA of two different white colonies of Ecoli, known as W1 and W2, and a blue colony called B. The DNA fragments from culture B is similar to the fragments produced by normal digested Puc19 in figure 1. There are two distinct DNA fragments of roughly 600 and 5000 base pairs in size shown on both W1 and W2. There is a faint additional band shown on W2. A Nanodrop is carried out in order to determine the DNA concentration of the culture samples B, W1 and W2. The DNA concentration in sample W2 was the highest, with 40.6 ng/uL, which is twice as high as the DNA concentration of W1 and B.