Rabies Virus - Dissertation Example

Rabies virus is a non segmented RNA containing negative strand and belongs to the Lyssavirus genus and comes from the Rhabdoviridae family. Rabies virus has an envelope protein called G-protein that protects and is responsible for the binding of the virus to the target cells. The use of rabies virus as vaccine for HIV and other viral diseases has brought in their efficiency to acts as pseudo titers. The level of expression of the pseudo titers in the HIV -1 is very less using the pl 18 plasmid containing RV277 glycoprotein gene. The expression level of the gene can be increased by the use of different plasmids. So in order to increase e the expression level of the Rabies virus, the RV277 glycoprotein gene was inserted into the pCAGGS plasmid using the molecular biology techniques such as plasmid DNA isolation, increasing the concentration of the insert DNA using the PCR, restriction digestion of the insert DNA and the plasmid pCAGGS vector, ligation of the insert and the vector using t4 DNA ligase and bacterial transformation of the ligated DNA in E.coli through the heat shock method. The transformed Bacterial cells were found to have positive results indicating that the transformation has worked. The future work of this project includes identification of the gene and further analysis of the effect of transformation of the RV 277 glycoprotein gene. The use of different expression plasmids to increase pseudovirus titres using the glycoprotein for rabies virus RV277 Introduction: Rabies virus is a prototypic and neurotypic member of the Rhabdoviridae family. It is present within the Lyssavirus genus. This virus consists of a 12 Kilo base pair RNA strand which is a negative strand. There are coding for five proteins which are very essential for viral replication and particle formation. This viral RNA is enclosed inside the viral nucleoprotein and has phosphoprotein enclosed in it. The RNA genome contains 2-3% RNA, 67 – 74% proteins, 3% carbohydrates and 20 – 26 % lipids. (Shors 2011). The glycoproteins of the rabies virus are important immunologically as they are capable of reacting very well with the viral particles. It induces and reacts with the viral neutral particles. The surface glycoproteins are responsible for the attachment of the virus to the receptors of the host. They are also responsible for the membrane fusion of the host cell with the vector by the pH mediated fusion. (Shors 2011). The glycopotreins or the G-proteins present in the rabies virus has the ability to accept foreign genes without affecting the rabies viral genome and its functions. Vector based viruses are widely used for delivering the therapeutic genes into the primary cells. HIV viruses are independent of cell division and they are different from the other viruses. The HIV -1 DNA is generally maintained as an extra chromosomal viral DNA in the CD4+ cells and other cells present in the human body. (Reiser et al, 1996). Most of the DNA is reversible transcribed and this transcription is partial. Retro based vectors have the capacity to infect the non dividing cells. The non dividing cells include stem cells, post-mitotic neurons and hematopoietic progenitor cells. If the HIV -1 type is used as a vector system by deleting the viral envelope, then the possibility to use them fruitfully will increase. (Reiser et al. 1996). By inserting the rabies viral envelope gene G- type protein called glycoprotein into the HIV -1 genome , by deleting its own viral envelope gene, the applicability of the vector in primary , non dividing human cells can be determined. (Reiser et al. 1996). Many vectors were developed using the pseudotype vector system. Some scientists concentrated on the two –plasmid system and some on encoding the placental alkaline phosphatase into the nef coding region of the pseudo vectors. The titer value was close to 2 x 10^ 5 / ml. (Reiser et al. 1996). Some scientists worked on the pseudo typing of the VSV- G and HIV-1 vector system. The vectors designed were even capable of transducing the monocyte- derived macrophages of the human. Possible studies on the gene transfer to the terminally differentiated neurons were also done. Many scientists have developed vector system for use as pseudo titers for the analysis of HIV- 1 infection and some are of great success. (Reiser et al. 1996).The vector systems developed by akkina and co workers proved that they have an efficient vector system with high titer value. The production of viral capsids in the laboratory conditions with different viral genome results in the production of pseudovirus. This pseudovirus can be developed in many methods. The recombinant viral vector Semliki Forest virus and Vaccinia virus had Papillomaviral capsid expressed through infection. (Campo 2006). Co-expression systems were also practiced for the production of the infectious capsids. Most of the pseudo virions produced were of 8 kb length. This length resembles the length of the papilloma virus genome. Most of the plasmids are taken by the cells at very low efficiency. (Rossman and Rao 2012). For a particular construction of the vector system, the level of expression of the reporter gene is very important as they reflect the number of pseudovirus produced. Thus the level of reporter gene expression is very essential for the pseudovirus particles to look and behave like the virions. (Mochizuki et al. 1998). Studies have found that the GUS pseudovirus particles has brought in a convenient tool for the assay of the virus like particles with no nucelocapsid. (Gallagher 1992). Thus these pseudovirus particles paves a way for the analysis of the coat protein expression based on un coating step of infection and introduction of new gene on the encapsulated mRNA. Pseudovirus can be used for the analysis of which part of the virus is highly pathogenic or non- infectious region. (Chang 1992).The level of the coat protein expression indicates the level of infection. The combination of the human rhinovirus with the HIV-1 virus at the membrane proximal external region was used for the ELDKWA patients for the neutralization effect. The use of the broad level of reacting neutralizing antibody for the defense of HIV was aimed for the clearance of the cell- free virions and for the destruction o the infected cells. Though there are many neutralizing monoclonal antibodies, producing immunogens which can perform the cross reaction and neutralize the HIV responses are very effective. So to improve the potency and rate of neutralizing the viral genomes, the development of pseudo virus is essential. (Arnold et al. 2009). Plasmids are extra chromosomal DNA present in the bacterial species. They are double stranded DNA which forms circles with size ranging from 1 kb to 200kb (kilobase). (Arnold et al. 2009). Plasmids are very advantageous for the genetic engineering. Plasmids code for many antibiotic regions and they have the ability to accept the gene of interest. The transformation of our gene of interest into the plasmid is called recombination and the bacteria are called recombinant bacteria. Thus plasmids can be used as cloning vehicles or vectors. Restriction endonucleases are the bacterial enzymes that cleave the double stranded DBA at specific ends. There are three types of restriction endonucleases. Type I, type II and Type III. Type II restriction endonucleases are mainly used for the DNA cleavage at specific sites. Restriction endonucleases are present in many strains of bacteria and they are specific for each strain. (Arnold et al. 2009). These restriction endonucleases digest the foreign DNAs at specifc ends and thus safe-guarding the parent strain. These Restriction endonucleases require specific temperature, pH for maximum activity. The restriction enzymes are stored at 4°C to maintain their activity level. (Arnold et al. 2009). The enzymes are not only used for the joining and ligating fragments but also for the synthesis of the proteins and even for producing million copies of the DNA segments in invitro technique. This is possible with the help of PCR. Polymerase Chain Reaction (PCR) enables the researchers to produce million copies of DNA sequences in approximately 2 hours with the help of a DNA template, 2 primers that are complementary to the 3’ ends of the two sense and anti-sense DNA fragments, Taq DNA polymerase Enzyme, Deoxy nucleoside triphosphates, buffer solution and divalent cations. (Daniel 2008). The PCR is also called as thermocycler. There are four steps in the PCR that are reapeated until the desired concentration is reached. They are denaturation, annealing, extension and renaturation. (Daniel 2008). 1. In the denaturation step, the DNA to be amplified is denatured into single strand molecules. The denaturation of the DNA is usually done at the temperature of 95? C. Now the DNA is present as single stranded ones. 2. The second step is the annealing of the primers to the DNA fragments. This is achieved at a temperature of 50-60 ? C. The variation of the annealing temperature is due to the high concentration of the G+C content. (Daniel 2008). 3. The third step is the elongation/extension step. After the DNA has bind with the primer, the oligo nucleotide binds to DNA strand according to the specifications. The DNA polymerase does the polymerization reaction. The Taq DNA polymerase which is derived from the thermos aquatics bacterium extends the primers at a temperature of 70? C. (Daniel 2008). 4. The last step is the renaturation step. After the DNA strand is being generated with the help of the primers, the Single stranded molecules binds with each other to form the double stranded DNA. The DNA duplexes that are formed are once again denatured, and annealed with the primers, elongated with the help of the oligo nucleotides and renatured to form the DNA duplexes again. Agarose gel Electrophoresis is the standard method for the separation of the DNA fragments based on their molecular weight. DNA fragments move through the Agarose gel under the influence of the electrical power. As DNA is negatively charged, it moves towards the anode. The pore size of the Agarose separates the DNA fragments based on their size. Thus the DNA fragments get separated based on molecular size, the current applied, concentration of Agarose and the conformation of the DNA. A standard ladder is used to identify the size of the fragments. The Bands on the gel are visualized using the intercalating fluorescent dye, Ethidium bromide. When the Agarose gel dyed with EtBr is visualized in the ultra violet rays (UV rays), we can observe the bands. (Daniel 2008). This method is very efficient as it can detect the ds DNA concentration as low as 1 nanogram. (Daniel 2008). Two DNA fragments are ligated using the Bacteriophage T4 DNA ligase. T4 DNA ligase catalyses the binding of 3’hydroxyl end of one nucleotide and 5’ phosphate end of other nucleotide, using the phosphate- di- ester bond. (Arnold et al. 2009). T4 DNA ligase will ligate the blunt ends, overhanging and complementary and sticky ends. It is easier to stick the sticky ends than the blunt ends. This catalytic reaction requires Mg2+ and ATP as the cofactors. The amount of the Ligase enzyme required varies based on the nature of the filaments to be ligated, stability of the hydrogen bonds to be ligated, and temperature and concentration of the DNA fragments. (Arnold et al. 2009). E.coli is the main hosts for Bacterial research as it can uptake foreign plasmid DNA more efficiently than other bacteria. E.coli and its plasmid structure is well understood many other bacteria. This is the main reason for using E.coli for transformation. This uptake of the plasmid DNA is achieved at the mid log phase of the bacterial growth. E. coli is sensitive to ampicillin, and this helps us to select the colonies form the non-transformed bacterial colonies. The recombinant plasmid contains ampicillin resistance gene. So if the recombinant plasmid is present in E.coli, the ampicillin resistant colonies alone will be formed. This will reduce the chance of contamination and easy screening of the cells. When E.coli is subjected to a sudden increase of temperature from 0°C to 42°C, the heat shock proteins are formed and they increase the membrane fluidity to enable the plasmids to get inserted into the E.coli. (Arnold et al. 2009). Methodology: Isolation of the Pi 18 plasmid DNA containing the RV277 Glycoprotein gene: The plasmid containing the RV277 glycoprotein was taken and the plasmid DNA was obtained using the alkaline lysis method and purified by ethanol precipitation and RNAse method. 1. The culture was grown overnight and 2 ml of the culture containing the plasmid Pl 18 was taken. 2. 1.5 ml of the stock culture was added to the microfuge tube. 3. The microfuge tube was centrifuged at 10000 rpm for 30 seconds. 4. The supernatant was poured off and the bacterial pellet was collected. 5. The pellet was re-suspended in the 100 micro liters of Re-suspension buffer. (Cold solution I). 6. The solution was vortexed to enable complete mixing for 2 minutes and the bacteria gets fully re-suspended. Vortexer is the simplest step to mix all the contents in the microfuge tube. 7. 200 micro liters of denaturing buffer (Solution II) was added to the tube and the tube was inverted and the contents inside the tube were mixed slowly. The solution was allowed to become clear and thicker. In this step the proteins and DNA were denatured. 8. The solution was incubated on ice for 5 minutes. 9. 150 micro liters of re-naturing solution (Solution III) was added to the tube. 10. The tube was inverted and mixed slowly. The proteins and DNA present in the tube were precipitated. 11. The solution was again kept on ice for 5 minutes. 12. The tube was centrifuged at 12000 rpm for 5 minutes. 13. The supernatant was transferred into a new tube. 14. To the supernatant, 700 micro liters of 100% cold ethanol was added and the plasmid DNA gets precipitated. 15. The supernatant was poured out and the pellet was washed with 70% ethanol. 16. The pellet was dried in air for 30 minutes and re-suspended with 50 micro liters of TE buffer. Purification of the insert DNA by PCR: The insert DNA (RV277 glycoprotein gene) was of very little in quantity. Increasing the quantity of insert DNA will help us to perform the experiment with ease. Polymerase chain reaction is carried out to increase the concentration of the insert DNA (RV277 glycoprotein gene). Polymerase chain reaction (PCR) increases the number of the insert DNA by many folds. This can be confirmed by the agarose gel electrophoresis. 1. The insert DNA (RV277 glycoprotein gene) was taken and to it the forward and reverse primer were added to it. The Forward Primer sequence is: 5’ GAG CGA GGT ACC TCC ACC ATG GTT CTT CAG GTT CTT TT 3’ The Reverse primer sequence is: 5’TCGAG TCA CAG TCT GGT CTC ACC 3’. 2. Tag DNA polymerase enzyme, phosphates buffer, insert DNA, forward and reverse primers, nucleotides and distilled and de mineralised water are added to the PCR tube and the DNA was amplified. 3. The amplified DNA thus obtained was then run in the gel electrophoresis and was observed under the UV light. The anticipated size of the insert DNA was 1.6kb. 4. 1 kb DNA ladder was added to the gel and the Insert DNA was identified. 5. The insert DNA was then isolated using the QIAquick PCR Purification Kit. Restriction Digestion of Plasmid DNA: The insert DNA and the plasmid were cut by the same restriction enzymes at specific sites for the ligation reaction in two different test tubes. 1. The pCAGGS plasmid and RV277 glycoprotein gene were restriction digested. 2. Kpn1 and Xho1 were the restriction enzymes chosen for the digestion of the pCAGGS plasmid and RV277 glycoprotein gene. 3. Two eppandrof tubes were taken and the restriction digestion reactions were carried out in each tube as follows. 4. For the first tube : 1 µg of amplified DNA was added to the eppandrof tube and to this 1 µl of the restriction enzymes Kpn1 and Xho 1 each were added . 3 µl of the buffer ( usually 10 x concentration ) was added. The volume was made upto 30 µl. 5. Similarly for the second tube : 1 µg of plasmid pCAGGS DNA was added to the eppandrof tube and to this 1 µl of the restriction enzymes Kpn1 and Xho 1 each were added . 3 µl of the buffer (usually 10 x concentration) was added. The volume was made upto 30 µl. 6. Both the tubes were gently mixed by pipetting. 7. The tubes were then incubated at 37 ° C for 1 hour. 8. The digested mixture is then visualized after performing the gel electrophoresis using the 1kb DNA ladder. Isolation of the insert (REs digested PCR product of RV277 glycoprotein gene) and recipient plasmid (REs digested pCAGGS vector) by gel-purification: 1. The Restriction digested PCR product of RV277 glycoportein gene was run on an agarose gel electrophoresis and the 1.6 kb product was cut and purified. 2. Similarly the Restriction digested pCAGGS vector was also run on the same agarose gel and the 4kb product was cut and purified from the gel and the concentration of both the insert and the recipient plasmid were identified. Ligation of the insert DNA (RV277 Glycoprotein gene) into the recipient vector (pCAGGS vector): 1. In an eppendrof tube, the following DNA Ligation mixture was prepared: 25ng of pCAGGS vector DNA 75ng of insert RV277 glycoprotein DNA. Ligase buffer ( 10 µl of 10 x buffer) 1 µl of T4 DNA ligase enzyme. Triple distilled water was added to make the volume upto 10 µl. 2. The mixture was incubated at room temperature for 2 hours. 3. The legated product and the control (pCAGGS with no insert) were run on the same agarose gel electrophoresis and the ligation was confirmed. Transformation: 1. The competent cells (Top 10 E.coli bacteria) were taken from the ultra freezer and thawed on ice for 30 minutes. 2. The agar plates were then prepared with the antibiotic ampicillin. 3. 5 µl of the ligated sample was transferred in to 50 µl of the competent cells in a microfuge tube and the mixture was gently mixed by flicking the bottom of the tube several times using the finger. 4. The competent cell mixture ( ligated DNA ( Insert (RV277 G gene)+ pCAGGS) + Top 10 E.coli bacteria ) was kept on ice for 30 minutes. 5. The transformation technique practiced was heat shock method. 6. The competent cell mixture was kept in the water bath at 42° C for 45 seconds. 7. The tubes were taken immediately and kept on the ice bath for 2 minutes. 8. To these tubes 500µl of LB broth (Luria Bertani broth) without antibiotic was added and grew at 37° C in the shaking incubator for 45 minutes. 9. The transformed cells were then plated in the LB agar plates with ampicillin and incubated at 37° C overnight. 10. Similarly the 2 control plates were prepared. One plate containing pCAGGS with no insert and the other plate with undigested pCAGGS plasmid. 11. The growth of the colonies in the plates was observed. Result: The DNA was extracted from the culture. The method used for the obtaining the plasmid DNA was alkaline lysis/ ethanol method. The plasmid DNA obtained in this method is used for the transformation process. The plasmid DNA may contain contaminants along with them such as RNA and small proteins. They are cleaved by using the RNAse enzyme and second precipitation using propranol. The plasmid DNa is confirmed by the agarose gel electrophoresis. Agarose gel electrophoresis is the method in which the DNA are separated based on their molecular weight and size. The insert DNA concentration was very less. So in order to increase the concentration of the insert DNA RV277 glycoprotein gene, polymerase chain reaction was performed. The Insert DNA was added with the forward and reverse primer and multiplication was performed using the tag DNA polymerase enzyme. At the end of many cycles, the PCR product obtained was found to have higher copies of the insert DNA. The PCR had increased the concentration of the Insert DNA. This was confirmed by the thicker band obtained in the gel corresponding to the DNA ladder in kb. Restriction digestion of the insert DNA (RV277 glycoprotein gene) and the plasmid pCAGGS was performed using the enzymes Kpn1 and Xho 1. Restriction digestion is the process of cutting the DNA strands at specific sites. Since these enzymes are specific in nature, they cut the DNA only when the required DNA strand is present. Xho1 and Kpn1 produce sticky ends and hence the process of ligation will be easier. Ligation is the process of joining together two DNA fragments. The ends were sticky ends and hence the ligation was very quick and perfect. The components were of equimolar and the ligation reaction was good. The insert and the plasmid DNA were borrowed from the other group and instructor for ligation reaction. The efficiency of the ligation can be checked using the Agarose gel electrophoresis. The ligation is perfect as can be seen from the Agarose gel electrophoresis. The ligation has occurred and this was confirmed by the thick single band present in the gel. Significant amount of colonies were found in all the plates. Colonies were found in the control 1 containing the undigested pCAGGS, control 2 plate with vector pCAGGs alone and in the plate with insert and vector (PCAGGS). Discussion: Rabies virus based vectors for Human immunodeficiency virus type 1 are very promising since they have very less pathogenicity and can best act as a vaccine. Rabies virus (RV) pathogenicity was studied for many years. The researches being conducted for the past 100 years have resulted in a wide range of variants. These variants range from highly pathogenic to non pathogenic. The pathogenesis of RV lies in two proteins P and G. The interaction between the RV P and the LC8 (cytoplasmic light chain) results in the production of partial pathogenesis by causing retrograde transport to the CNS (central nervous system). (McGettigan et al. 2003). Similarly large evidences show that G protein is only responsible for neutralizing the antibodies and can act as the major contributor for pathogenicity in the virus. The deletion of the RV G has proved the reduction of viral titer and the spread of Rabies virus. The use of rabies virus as a potential pseudo virus titer and effective vehicle vaccine was found to be possible. (McGettigan et al. 2003). The HIV infected individual’s develop immune deficiency and later AIDS. Though there are more therapies for the HIV –1 infection, the need for a proper protective vaccine is still under development. Most of the vaccines used so far contain either killed viruses or attenuated viruses. These types of vaccines are of no use to HIV – 1 patient because killed vaccines produce only very less amount of neutralizing antibody response. (McGettigan et al. 2003). Attenuated vaccines are also not possible because a similar virus called Simian immune deficiency virus was found to cause AIDS like diseases in monkeys. So the use of new technologies for the development of HIV -1 vaccine is necessary. The uses of recombinant viral vectors for the protection against the harmful diseases have shown fruitful results in the past few years. Rabies virus is a negative stranded RNA virus that has five structural proteins that are very simple and perfect for the modular genome organization. Using a non pathogenic RV vaccine based vector for the analysis and treatment of HIV – 1, has shown great results. This type of approach is better than other methods. The alteration of the genetic structure is easy for the RNA based viruses. Rabies virus is more stable and keeps the large strand of foreign DNA with it. Above all, the most important thing to consider is no recombination of the genome to the host cell. The serum positivity is negligible for the RV in the human population and the vector based RV will not interfere with the HIV- 1 genome. (McGettigan et al. 2003). The expression level of RV is very high in the titers for many cell lines. This results in the longer expression of the cells to the HIV-1 genes. The recovery of the rabies viruses of the Rhabdo viruses which are very infectious from the cDNA construct has given hope for the genetic manipulation. By inserting the foreign genes such as vesicular stomatitis virus and paramyxovirus into the rabies viral genome and recovering it from them has proved to be very fruitful. (McGettigan et al. 2003). These viral proteins are needed for the formation of the active transcription complex. This indicates that these complexes are highly active and flexible. The growth and expression of the other genes in the rabies virus did not affect the ability of the rabies virus. Thus it was concluded that rabies virus has the ability to accept a wide range of foreign genes in it with the ability to express them successfully along with the normal functions of the rabies virus. When a study was conducted to estimate the ability of the virus to express a heterologous protein, it was identified that they were able to express the entire foreign unit gene with the complete recovery of the rabies virus. Thus a variety of the DNA viruses and many positive strand RNA viruses have been found to express the heterogeneous foreign gene. This has created a greater interest for the clinical and basic research for the potential use of the gene. The simple modular organization of the genomes and the better efficiency for engineering the genome and the formation of the protein complex of the genome helps a lot for the genetic engineering approach. Many studies have confirmed that mini genomes can be successfully inserted in to the rabies viral genome. The basic strategy of expression present in rhabdoviruses, paramyxoviruses and filoviruses are very common and this is the main reason for them to encode and express a given gene. (McGettigan et al. 2003). This success in the rabies virus is possible only because of the 5 viral proteins present in the rabies virus. These genes are so intact that the presence of an additional gene will not interfere in their biological activities. This stability enables the rabies virus to act as a vector. The recombinant rabies virus was able to obtain the non essential regions of the virus indicates that they are very intact and more stable. This is another reason for using the rabies virus as vector. (Mebatson et al. 1996). Pseudo viruses are the viruses that carry the core and genome of one virus with the envelope of the second virus. This type of pseudo viruses are used for the entry of the cell and for the receptor identification processes. The viral neutralization by the antibodies is the most important application of pseudo viruses. Retroviral pseudo types are the commonly used vectors for the gene therapy. The retroviral pseudo types with the rabies envelope were used to function as the neurotropic gene vectors. Since the envelopes are very sensitive and specific, many assays can be performed for the identification of the pathogens. The gene coding for the rabies virus is encoded in the pCAGGS plasmid and inserted into the lentiviruses. (McGettigan et al. 2003). The presence of the G- protein from the rabies virus helps to play the cross- neutralization step. The glycoprotein of the rabies virus is responsible for the expression of the native proteins on the wild type. Many live RABV virus particles are found to allow the entry of the particles and are neutralized by the anti –RABV sera. (McGettigan et al. 2003). Using the Antibody to detect the rabies viral G protein, the presence of these proteins in other cells was also determined. The glycoprotein of the rabies virus dominates the pseudo type particle formed. The glycoprotein was found to incorporate with equal efficiency in the pseudo types. Modification of the HIV virus is possible if the assay was developed using the replica and incompetent particles. This was tried in this project. The RV277 glycoprotein gene was increased in the concentration by the use of the PCR technique. The concentration of the insert DNA RV277 glycoportein gene was confirmed by performing the agarose gel electrophoresis. (McGettigan et al. 2003). The second important step was the isolation ofhte plasmid DNA pCAGGS. This was done by using the alkaline lysis / ethanol method. Alkaline lysis solution ((i.e.) the denaturing buffer) was added first to denature the proteins and rupture the cell wall enabling the bacteria to lyse and release the cell components in to the solution. Second the renaturing solution as added to the medium. This brings back the pH and causing the protein and DNA to precipitate and leaves the bacteria in the solution. The renaturing solution contains ethanol which precipitates the protein and DNA. the supernatant was then removed containing the plasmid DNA. (McGettigan et al. 2003). The plasmid DNA was then purified from RNA by adding the RNAse enzyme. The plasmid DNA thus obtained is now ready for fusion. After the isolation of the pCAGGS vector and the PCR product both were restriction digested using the sticky end restriction enzymes Xho 1 and Kpn 1. 1 micro liters of the sample was subjected to restriction digestion separately in the two separate test tubes. After the restriction digestion, ligation of the insert and the Vector DNA was made by mixing equal amount of insert RV277 glycoprotein gene and pCAGGS plasmid and ligating in the presence of the enzyme DNA ligase. The recombinant plasmid was thus produced. This recombinant plasmid was then inserted into a suitable host for the multiplication and to ensure perfect recombination of the plasmid. Bacterial transformation by heat shock method was practiced. The bacteria Escherichia coli were used for the transformation process. Here the E.coli which is sensitive to ampicillin antibiotic was used. by keeping the ligated DNA and the bacterial culture together in the ice followed by a heat shock for 30 seconds at 42 degree Celsius and then immediately bringing them back t o the room temperature enables the pores in the cell wall to open and the ligated plasmid will enter inside the cell. The immediate cooling will close the pores in the cell wall and thus the bacteria become transformed. The bacterial transformation was confirmed by using two controls. One control contains only the ligated DNA and the second control contains the native pCAGGS plasmid. It was observed that all the three plates produced some colonies. The growth of the E.coli in the ampicillin resistant LB agar plate confirms that the transformation has occurred. 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