Molecular Cloning and Purification of Green Fluorescent Protein - Lab Report Example

Molecular Cloning and Purification of Green Flourescent Protein (GFP) Adapted from "BioRad Biotechnology Explorer: pGLO Bacterial Transformation kit" (#166-0003EDU) ABSTRACT: The experiment aims at cloning and purification of the green fluorescent protein (GFP) protein using molecular techniques like transformation, restriction digestion and vertical gel electrophoresis. Briefly, the pGLO plasmid incorporated with the GFP gene, regulated by the arabinose inducible promoter was transformed into E.coli strain HB101. To confirm the success of transformation, the recombinant DNA was isolated and purified from the transformants followed by digestion using HIND III /EcoRV. The GFP protein was then purified from the transformants using hydrophobic interaction chromatography (HIC) and its purity confirmed using immunoblotting and SDS-Page. 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. RESULTS AND DISCUSSION: 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. The bacteria was then plated onto four sets of defined media as suggested in the protocol. The plates 1-LB and 2-LB+Amp were streaked with E.coli minus the pGLO plasmid and 3-LB+Amp and 4-LB+Amp+Ara with E.coli along with the pGLO. 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 the plasmid DNA and digestion with this enzyme yielded a single band of 5kb as observed in lane C of the gel, thus indicating the size of the plasmid to be 5kb. HindIII digestion of the plasmid yielded a large fragment of 3 kb and a smaller one of 2 kb. Double digestion revealed the presence of three fragments of 3kb, 1.5kb and 500bp, in accordance with the predicted sizes, indicating that the first HindIII site could be present between the EcoRV site and the second HindIII site, as the 3 kb fragment yielded by HindIII digestion is intact, while the sizes of the other fragment has been significantly altered with respect to both EcoRV and HindIII. The Hydrophobic interaction chromatography technique, used to isolate the GFP protein led to the recovery of approximately 82.7% of the total protein (Table-1), making it a reliable system of protein isolation. The cells were lysed and the proteins released into the supernatant, also called the 'Crude Lysate', which fluoresces when observed under U.V, due to the presence of GFP along with other cellular proteins. The SDS-Page analysis revealed the presence of the GFP protein in the cell lysate obtained from transformed cells, indicating a successful insertion and expression of the plasmid DNA. The SDS-Page gel upon coomassie staining (Figure 2) revealed the GFP protein along with various other cellular proteins or impurities which emphasizes on the requirement of better processing techniques, to avoid ambiguity in interpretation of results. The immunoblotting analysis confirmed the presence of the GFP protein upon treating with mouse anti-GFP monoclonal antibody. PLEASE INCLUDE BRADFORD RESULT. CONCLUSION: The data indicates that GFP protein was successfully cloned and purified using simple molecular biology techniques. The same protocol and vector may be used to clone and isolate a gene of interest from any other biological systems, using GFP as a reporter. Table 1: 1 2 3 4 5 Figure 2: Coomassie stained 10% SDS-Polyacrylamide gel showing the presence of GFP Protein in crude lysate along with impurities in lane 2 and in the eluted fragment in lane 5. Lane 1: Molecular marker. Lane 2: GFP-CL, crude lysate Lane 3: HIC 2, lysate fraction treated with binding buffer Lane 4: HIC 3, lysate fraction treated with wash buffer Lane 5: HIC green fluoro fraction upon elution. Read More