Health and Life Sciences Faculty Course Title: Biological and Forensic Science Module code: 216 BMS Module Title: Molecular Genetics Overview of the Recombinant DNA technology- the process of subcloning a foreign gene into the plasmid vector puc19 Ciobanu Maria Alice SID: 3395606 Word count: 1617
Overview of the Recombinant DNA technology- the process of subcloning a foreign gene into the plasmid vector puc19 Introduction The term gene cloning refers to a wide variety of techniques that makes it possible to manipulate DNA in order to return it to living organisms where it can function normally. Essentially, it involves isolating a piece of DNA from an organism and introducing it into a cloning host, for example bacterium Escherichia Coli which grows and divides rapidly. It is therefore possible to study the cloned DNA or produce the protein encoded by the gene.the cdna may be inserted into vectors and then cloned. The choice of vector represents the most important consideration in molecular cloning experiments. (Krebs et all, 2010) Many bacteria contain an extra chromosomal element of DNA known as a plasmid. This is a relatively small, covalently closed circular molecule, which carries genes for antibiotic resistance, conjugation or the metabolism of unusual substrates. One of the most notable plasmids, termed puc19 was widely adopted for cloning of small DNA fragments in E.coli. It is an antibiotic resistance gene for ampicillin and contains an origin of replication enabling the vector to replicate in E.coli. (Lodge et all; 2007) Plasmid vectors permit the identification of recombinant clones by looking for insertional inactivation of either antibiotic resistance genes or lacz. However, potential recombinant clones still need to be analyzed. This involves purifying the plasmid DNA from individual clones, cutting it with restriction enzymes in order to analyze the size of the fragments produced. (Krebs et all, 2010) Restriction endonucleases recognize certain DNA sequences and cleave them in a defined pattern. Covalent joining of ends on each of the two strands may be brought about by the enzyme DNA ligase, which enables the construction of recombinant DNA fragments. However, it is often convenient to cut a vector with two restriction endonucleases which do not produce complementary overhanging ends (described as sticky ends and are generated by cleavage). EcoRI is one of the most commonly
used restriction enzyme and it is produced by the bacterium E. coli strain RY13. (Walker, J.M., Rapley, R. ;2000) The next step in cloning a gene is to find a way of joining the DNA molecules together in a new combination (termed as recombinant). The most common used DNA ligase is a protein produced by a bacteriophage known as T4. In the energy consuming process of ligation, DNA ligase catalyzes the formation of a covalent phosphodiester bond between the 5 phosphate on one DNA strand and a 3 hydroxyl on another. (Krebs et all, 2010) The final step is described as the transformation which involves the introduction of the new recombinant plasmid into E. coli. For the DNA to get into the bacterial cell, a selection of the cells containing the plasmid is necessary. Considering that E.coli is not naturally capable for transformation, its cells need to be treated by chemical treatment and electroporation in order to enable them to take up DNA. (Lodge et all; 2007) This laboratory report is based on three practical sessions and its aim is to transfer a fungal gene (termed CIH-1)from a plasmid vector (called pbk-cmv) into a different plasmid vector (known as puc19) through the use of a number of techniques and procedures as restriction endonuclease digestion of DNA, analysis of DNA fragments by agarose gel electrophoresis, ligation of DNA fragments into a vector, introduction of the ligated DNA into host bacteria by transformation, selection of colonies containing recombinant vector molecules and isolation and analysis of recombinant plasmids. (Coventry University; 2011) Methods: The experiments were carried out as described in the schedule. (Laboratory Schedule, Coventry, 2011)
Results In order to clone DNA, it needs to be cut up in a precise and repeatable way by using enzymes. Therefore, the foreign gene (CIH-1) needs to be cut out of the pbk-cmv with the restriction endonucleases EcoR1 and Xbal, same as the puc19. (Coventry University; 2011) To check if the restriction digestion has been successful, gel electrophoresis is used to measure the size of the fragments generated as it can been seen in Figure 1 below: Figure 1. Agarose gel electrophoresis of the restriction digest plasmids pbk- CMV and puc19 It can be observed that the nucleic acids migrated in gel describing a linear movement of the DNA fragments. The DNA is visualised by staining with ethidium bromide, which fluoresces under ultraviolet light. From this image, the measurement of the distance moved by each DNA size markers from the well can be determined using a ruler. The results concluded have been recorded in Table 1 below. Table 1. Distance moved by DNA fragments from the well DNA fragments size (base pairs) Distance moved from well (mm) 5000 10 3000 12 2000 14 1000 19 500 23
It can be observed that the distance that DNA fragments have migrated from the well is proportional to the size of the DNA fragment, with small fragments moving faster than large ones, describing a variation from 10 to 23 mm distance travelled from well. Furthermore, the size of fragments needs to be determined. A very useful tool is to plot a graph of log marker size against distance travelled in the gell. A calibration curve will be produced and used afterwards to calculate the size of puc19 and pbk- CMV DNA fragments. A representation of it can be observed in Figure 2 Figure 2. Representation of the measurement of the distance moved by DNA size markers The results established from the plotted graph have been recorded in the table below. Taking into consideration that the recombinant plasmids have been cut with EcoR1, two fragments have been generated: puc19 which varies in size between 600 bp and 3800. On the other hand, when Xbal is used to cut the plasmid, only one band occurs: pbk-cmv which has a length of 3100 bp. (Table 2) Table 2. Distance moved by restriction plasmids from the well Distance moved Size (bp) (mm) puc19 9 3800 20 600 pbk-cmv 12 3100
Following the ligation process, DNA needs to be introduced into the host cell. Therefore, the antibiotic ampicillin is added to the L-agar plates in the experiment in order to select for bacteria which have taken up the plasmid puc19. Also, a mixture of X-gal and IPTG are added. As it is an artificial substrate for β-galactosidase, X-gal produce a blue product. On the other hand, IPTG is an artificial inducer of the lac operon, stimulating its transcription (Coventry University; 2011). As a result, bacterial colonies which express β-galactosidase will appear blue, while the non-producing ones will be white. After counting the colonies, the results were recorded in Table 3 below. Table 3. Results determined from the transformation plates Sample Dilution No. blue colonies No. white colonies Plate 1 Plate 2 Mean Plate 1 Plate 2 Mean Compotent cells 10-6 0 0 0 257 190 223.5 10-7 0 0 0 63 51 57 10-8 0 0 0 2 6 4 Tranformation negative control (tube 3) Tranformation positive control (tube 2) Tranformation Ligation (tube 1) None 0 0 0 0 0 0 None 162 94 128 0 0 0 10-1 22 41 31.5 0 0 0 10-2 1 2 1.5 0 0 0 None 15 18 15.5 23 23 23 10-1 1 0 0.5 0 0 0 The Figure 3 represents the agarose gel electrophoresis of B1, W1 and W2 restricted plasmids. As it can be observed, W1 and W2 contain recombinant DNA, therefore they form 2 DNA fragments in the gel. Conversely, puc19 is nonrecombinant DNA, so just a single line is shown.
Figure 3. Agarose gel electrophoresis of B1, W1 and W2 restricted plasmids Measurements of distance travelled by the DNA fragments in the marker track are documented below. (Table 4) Table 4. Distance travelled by DNA fragments in the marker track DNA fragment size (base pairs) Distance moved from well (mm) 5000 6 3000 8 2000 9 1000 13 500 17 In order to determine the DNA fragments size, a calibration curve was plotted. It can be visualised in Figure 4 below.
Figure 4. Representation of the measurement of the distance travelled by the DNA fragments The estimation of the size of bands in the restriction digest is recorded in Table 5 and its based on measurements made on the calibration curve above. Table 5. Distance travelled by B1, W1 and W2 in the marker track Sample Distance moved Size (base pairs) B1 7 3300 W1 7 3300 14 550 W2 7 3300 14 550 It can be observed that the plasmids moved proportional to their length, resulting in the same number of base pairs. Due to the fact that W1 and W2 contain cdna, they produced 2 DNA fragments with a size of approximately 500 bp.
Discussion In the first experiment the fungal gene, CIH-1 which is isolated from the fungus Colletrotrichum lindemuthianum needs to be inserted into pcu19.the CIH-1 cdna have been cloned in a plasmid vector called pbk-cmv. In order to clone DNA, it needs to be cut up in a precise and repeatable way by using enzymes. Therefore, the foreign gene needs to be cut out of the pbk-cmv with the restriction endonucleases EcoR1 and Xbal, same as the puc19. Restriction endonucleases recognize certain DNA sequences which are polindromic, usually 4-6 base-pairs (bp) in length, and cleave them in a defined pattern. This means that the nucleotide sequence reading is the same in both directions on each strand. Usually they leave a flush (blunt ended) or staggered fragment when cleaved, depending on the enzyme. (Krebs et all, 2010) After inactivating the restriction enzymes, the plasmid and restriction enzyme fragments are mixed in the presence of T4 DNA ligase. In this experiment, throughout the ligation reaction the digested pbk-cmv and puc19 were mixed together. As a result, the foreign gene (CIH-1) from pbk-cmv is ligated into the MCS of pcu19. However, the desired outcome from the cloning experiment is that one vector molecule to be joined to one of the genomic DNA fragments in order to circularize and form a new recombinant molecule. The last step in gene cloning is the introduction of the recombinant plasmid into E. coli. During transformation, the DNA associated with the lipopolysaccharide on the outer surface of the competent cells in order to uptake the DNA. (Lodge et all; 2007) The most popular restriction sites are concentrated into a region called the multiple cloning site (MCS) which is located within the gene lac Z. Nevertheless, the MCS is part of a gene in its own right and codes for a portion of polypeptide called β- galactosidase which is caused by adding an inducer known as IPTG (isopropyl-β-dthiogalactopyranoside). The functional enzyme is able to hydrolyse a colourless substance named X-gal (5-bromo-4-chloroindol-3-γl-β-galactopyranoside) into a blue insoluble material. When a disruption in the gene occurs through the insertion of a foreign fragment of DNA, a non-functional enzyme results which is incapable to perform hydrolysis of X-gal (Krebs et all, 2010). Moreover, X-gal is the artificial substrate used in this experiment and IPTG is the artificial inducer which takes care of the repressor gene and stops it from working. Hence, it is easy to detect the recombinant puc19 plasmid since it is white in the presence of X-gal, whereas a non-recombinant puc19 plasmid will be blue as the gene is not disrupted, therefore
fully functional and expressing β-galactosidase activity. This impressing system, termed blue/white selection permits the initial identification of recombinants to be undertaken very rapidly. It is based on the lac Z gene and requires the use of special E.coli host strains which are naturally lac +. In fact, this represents one of the biochemical characteristics routinely used in the identification if E.coli. From Table 3 it can be observed that the number of white colonies overcomes the number of the blue ones. The white colonies are formed as a result of the insertion of DNA fragments into the multiple cloning sites of puc19 which interferes with lac Z. If the bacterial colonies have taken up the plasmid puc19 they are coloured in blue. (Walker, J.M., Rapley, R.; 2000) The final step is to prove that the inserted DNA fragment in puc19 generated in this experiment is in fact the fungal cdna molecule, CIH-1. To start with, parts of DNA molecules from two chromosomes differ from each other by a single base pair, which results in the absence of an EcoR1 site in one of the chromosomes. Upon digestion with EcoR1, the chromosome without the extra EcoR1 site produces a larger fragment than the other one. This difference is recognised using a probe that hybridises within the region encompassed by two flanking EcoR1 sites present in both molecules. A probe represents a molecule able to bind very specifically to other molecules, therefore it is used to identify the relevant clone among the undesired ones. Two different kinds of probes are recognized: antibodies and polynucleotides. (Sudbery, P. & Sudbery, I.; 2009) To conclude, significant improvements have been made at the molecular level. Many new and powerful ways for isolation, analysis and manipulation of nucleic acids have been discovered. The recently developed cloning strategies heralded a new and exciting era in the exploitation of DNA molecules. Gene cloning especially enabled numerous discoveries to be made and provided precious insights into gene structure, function and regulation, becoming not only an extremely useful tool but also an absolute requirement in the area of bioscience. (Strachan, T., Read, A.; 1999) List of references
Coventry University. (2011) Laboratory schedule for 216BMS Molecular Genetics DNA cloning Labs 1-3. Coventry: Coventry University Krebs, J.E., Goldstein, E.S., Kilpatrick, S.T. (2010) Lewin`s essential genes. 2 nd edition. London: Jones and Bartlett Publishers Lodge, J., Lund, P., Minchin, S. (2007) Gene cloning Principles and Applications. 1 st edition. Abingdon: Taylor & Francis Strachan, T., Read, A. (1999) Human molecular genetics. 2 nd Garland Science Sudbery, P., Sudbery, I. (2009) Human molecular genetics. 3 rd Benjamin Cummings edition. Oxford: edition. Essex: Walker, J.M., Rapley, R. (2000) Molecular biology and biotechnology. 4 th edition. Cambridge: The Royal Society of Chemistry