The gels were visualized by coomassie staining. The restriction digestion of recombinant DNA yielded the predicted band sizes upon electrophoresis, confirming the presence of the pGLO plasmid in the transformants. The presence of a single neat band at the expected size range upon immunoblotting indicates the successful isolation of the purified GFP protein.
Initially obtained from the jellyfish Aequorea victoria/Aequorea aequorea/Aequorea forskalea, the Green fluorescent protein (GFP) is composed of 238 amino acids with a molar mass of 26.9 kDa. Its typical three dimensional structure facilitates a specific set of cyclization reactions inside the protein giving rise to the tripeptide Ser65-Tyr66-Gly67 which forms the fluorophore, the fluorescent component of the protein, present on the alpha helix. This helix is actively shielded from the surrounding environment by the beta sheets, hence contributing to the use of the GFP gene as a reporter of gene expression or cellular protein localization.
In the present experiment, the GFP gene has been used to understand the mechanism of molecular cloning and subsequent protein purification procedures. The molecular cloning was carried out by transforming the pGLO plasmid vector with the GFP gene insert into the bacterial host E.coli strain HB101. ...
After appropriate incubation, the colonies were observed under normal light, followed by U.V light. Under normal light all the plates except plate with LB+Amp had growth
Lack of plasmid DNA on Plate with LB+Amp-pGLO might have rendered the colonies
susceptible to the antibiotic ampicillin, due to absence of genes required to deactivate ampicillin. Flourescence was observed in plates 3 and 4 under U.V light indicating the transformation of the plasmid DNA into the E.coli host. Plate 3, inspite of having ampicillin showed growth due to the presence of B-lactamase gene which can inactivate ampicillin. Plate 4 had the maximum number of colonies compared to the other plates.
This could be attributed to the addition of arabinose into the medium which selectively enhances the activity of the arabinose operon in which the GFP gene has been inserted, thus increasing the GFP protein which has the unique quality of fluorescence.
To confirm the insertion of the pGLO plasmid into the colonies on plates 3 and 4, the DNA from these colonies was purified using QAI kit method and subjected to restriction digestion using EcoRV and Hind III followed by electrophoresis, wherein an electric field was applied to the gel matrix. DNA molecules move towards the anode due to negativity of the charged phosphates along its backbone. The rate of migration of a particular DNA fragment is inversely proportional to its molecular weight; hence the fragments with the highest weight have the least mobility. Post electrophoresis, the ethidium bromide stained gel was visualized under UV light (Figure 1). A single restriction site specific to EcoRV is present on