DNA cloning technique
Cloning is the process of transferring a gene from its natural chromosomal to an autonomous vector so that to replicate. During the process of cloning, the DNA is transferred from the cells and manipulated in a test tube and the new DNA is consequently returned into cells (Lodege et al., 2007, p. 279). Since the genetic information of E. coli was initially well characterized, it has been the instrument of choice for DNA manipulation. One of the required combination vector and new DNA has been constructed in E. coli; the construct can be placed in other types of cells (Lodege et al., 2007, p. 282). There are two approaches to reproduce DNA; the Polymerase Chain reaction and the Cell-based process. This study will use the latter approach where the vector pTTQ18 will be used for expression. The vector also has to contain a very important gene, the antibiotic resistance gene. This study uses an ampicillin resistance gene to conduct the cloning process.
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Green fluorescent protein (GFP)
The Green Fluorescent Protein has been in existence for a long time probably over one hundred million years. This unique protein is found in a particular type of fish naturally, a jellyfish called the Aequorea Victoria, this protein is expressed in the photo organs and coded for by a single gene that has been isolated (Chalfie & Kainnzm, 2006, p. 1). The GFP gene can be inserted downstream of the promoter in any other gene from any other animal and be replicated. Because of the fluorescent properties of this gene, it has been the most commonly used reporter protein. An aequorin protein in jellyfish gives off blue light when it binds with calcium. The blue light is consequently absorbed by the GFP gene and in turn, gives off green light. The RNA polymerase is a useful enzyme in the process and it helps in binding the promoter area and induces transcription. If the GFP gene is well inserted in the plasmid correctly, it can be expressed in other organism like pigs and worms among others (Chalfie & Kainnzm, 2006, p. 3).
The GFP gene is helpful in observing when proteins are made and where they can be taken. The GFP gene is attached to the specific gene for the protein under observation. When that protein is made, it will contain GFP. With GFP fluorescence characteristics in play, the protein can be seen when light is shone and green light is given off (Chalfie & Kainnzm, 2006, p. 3). The GFP gene is for that reason a vital visual tag that assists in the observation and analysis of other genes’ expression.
The plasmid vector pTTQ18
In modern laboratories, researchers purify recombinant Taq DNA polymerase and assay for its pertinent role in the DNA. The E. coli will be used for purifying the recombinant protein (Society for General Microbiology, 2007, P. 161). The E. coli is used because it contains the pTTQ18 plasmid which in turn contains Taq DNA polymerase I gene that has been cloned behind a very strong and repressible promoter known as the Ptac promoter. This promoter comprises the 10- and 35- regions of the Trp operon promoter and LacO aspect from the lac operon. pTTQ18 also has the lac I gene and the marker gene for Ampicillin (Society for General Microbiology, 2007, P. 161). The reason for using this plasmid instead of using pGEM/Taq plasmid is that the pTTQ18 is a better option for expressing the gene as it has a very strong promoter hence very reliable.
The restriction enzymes
Restriction enzymes and plasmids form the basis of developing the cloning technique. The restriction enzymes have the ability to destroy infecting viral DNA without damaging the DNA of the host bacterium therefore the modification system works as a type of immune system for single bacterial strains safeguarding them from foreign DNA infection (Wong, 2007, p. 69).
The restriction enzymes form a junction as part of the restriction-modification structure. A matched modification can recognize and modify the nucleotide sequence that was recognized by the restriction enzyme, this is normally by methylation (Wong, 2007, p. 69). The methylated DNA is hence protected from cleavage by the restriction enzymes. The restriction enzymes otherwise called endonucleases can identify exact nucleotide sequences in DNA and cut both of them. There are two types of restriction enzymes namely class I and class II enzymes. Not all these enzymes cut the sequences at specific sites. Class, I enzymes include EcoB, EcoPI and Ecok while examples of class II enzymes include EcoR and PstI (Wong, 2007, p. 72).
Chalfie, M & Kainnzm, S. (2006). Green Fluorescent Protein: Properties, Applications, and Protocols, New York: John Wiley And Sons
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Lodege, J., Lund, P.A & Minchin, S. (2007). Gene Cloning: Principles And Applications, London: Taylor And Francis
Society for General Microbiology, (2007). Microbiology, Volume 153, Highwire Press
Wong, D.W.S., (2007). The ABCs of Gene Cloning, Boston: Birkhauser