Green Fluorescent Protein (GFP): Biology assignment essay
INTRODUCTION
The jellyfish Aequorea Victoria express a naturally green fluorescent protein called (GFP, about 12kD) .GFP contain a serine, tyrosine, glycine sequences whose side chains spontaneously cyclize to form a green fluorescing chromophore when illuminated with blue light .Using recombinant DNA technologies ,it is possible to make a DNA construct in which the coding sequence of GFP is fused to the coding sequence of a protein of interest .When introduced and expressed in in cells ,a GFP “tagged” protein is made in which a protein of interest is covalently linked to GFP as the part of the same polypeptide .Although GFP is a moderate size -protein ,the function of the protein of interest is often not changed by fusing it to GFP .This now allows one to visualise the GFP and hence the protein of interest .Not only can one immediately see the localisation of the GFP tagged protein ,but one can view its distribution in a living cells over times and thereby assess its dynamics or track its localization following a various cell treatment. The simple idea of tagging specific protein with GFP has revolutionized cell biology and led to development of many different fluorescent protein (Lodish et al 2013).It is often used to identify transformed cells, measure gene expression, and label and locate fusion proteins and study intracellular protein traffic, etc.(Tsien,1998).
Gene cloning
Molecular cloning involves isolating a DNA sequence and to amplify it in prokaryotic or eukaryotic cells. A DNA fragment can be incorporated into a circular DNA plasmid vector to construct a recombinant DNA molecule, which will be taken up by these cells to allow propagation. It allows the structure, function and regulation of the gene of interest to be understood for different uses, i.e. genetic screening, therapeutic development, etc. (Reed et al 2013).
In this study, bacterial plasmid pUC18/19, a prokaryotic vector where GFP genes to be over-expressed were placed downstream of the lactose-inducible trp-lac (tac) promoter and in-frame with part of the lacZ gene. The pUC vectors were introduced in the early 1980s and are based on fragments of DNA derived from naturally occurring Escherichia coli plasmids figure 2 shows the structure of the pUC18 plasmid .The pUC19 vector is identical except its genes are organised in the opposite direction .The pUC18 vectors have an origin of replication allowing them to be replicated in high copy numbers. They contains the gene for β -lactamase ,which confer resistant to the antibiotic ,(ampr) and allows the transformed cell which have acquired the plasmid ,to be identified .Additionally ,pUC18/19 also contain a portion of the E.coli lac operon ,which allows cells containing the plasmid to synthesis the amino terminal portion of β-galactosidase .pUC vector may be used with appropriate host cells that have been modified to express the gene for only the carboxy terminal portion of the enzyme .In the presence of suitable inducer, for example isopropylthio-β-galactosidase (IPTG),those cells containing a pUC plasmid will induce the two fragments of enzyme and possess β-glactosidase activity .Such cells are able to hydrolyse the artificial substrate 5-bromo-4-choloro-3-indolyl-β-galactoside (X- gal) to form a blue colour product .Thus, colony of the cells will appear blue when grown on nutrient agar containing the IPTG and X-gal.However, pUC18 also contains multiple cloning site near the lac gene ,which contains the recognition sites for a number of restriction enzyme (REs).The formation of a recombinant plasmid means the inserted fragments of DNA in this region prevent the formation an active β-galactosidase .Hence transformed cells possessing the recombinant DNA will be unable to hydolyse X-gal and their colonies will appear white and are so easily identified (Glencross et al ,2010).The steps involved in performing recombinant protein expression can be seen in figure 1
The aim of this study was to demonstrate molecular cloning by inserting the gene for GFP into bacterial plasmid vector pUC18/19 and transformed the recombinant vector into competent E.coli cells in order to express our protein of interest GFP and produce a green colour under microscope.
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