Transformation of E.coli with Plasmid DNA & GFP Purification - Essay Example

Introduction In biotechnology, the use of bacteria to express a recombinant protein is a routine practice especially during cloning process. Among notable bacteria used is E.coli, in the presence of a plasmid vector. The E coli are especially used in cloning and amplifying process whereby they produce various DNA pieces carried in a plasmid vector. The plasmids are found naturally and move in relation to the activities of the bacteria; such as when the bacteria divide making them move in vertical direction and in horizontal direction when DNA is taken up from the environment (Stone & Murphy, 2014, 8). Green Fluorescent Protein (GFP) is one of the recombinant genes usually produced and carried by the plasmid known as pGLO. The pGLO aids in transformation of E.coli and also subsequent expression of GFP. The plasmid contains three genes: Bla that is used in transformation of the bacteria, araC that is responsible for GFP expression and GFP that produce the characteristic fluorescent green color (Aune & Aachmann, F 2010, 1310). The following map illustrates the function of the pGLO in the extraction of GFP: Ori represents origin of replication whereby the plasmid is allowed to replicate independent of the bacterial DNA. Bla represents the ampicillin resistant gene which aids in secretion of beta-lactamase used in hydrolysis of antibiotics After the expression of the protein, need arises to purify it from the many proteins found in E.coli. Hydrophobic Interaction Chromatography comes out as the widely used process given its effectiveness in purifying a single recombinant protein from multiple gene products (Stone & Murphy, 2014, 8). The column chromatography method makes use of the relationship between protein-ligand interaction and salt concentration. At high salt concentration, the interaction is strengthened and the protein remains bound to the column. However, as the concentration decreases so does the interactions weakens thereby allowing GFP to flow down through the column. The major purpose of this experiment is to study the transformation of E.coli, to express GFP protein, and the isolation of GFP, per se. The bacterial transformation will be achieved by using pGLO whereby the DNA is incubated with the bacteria in a solution of CaCl2 to allow the DNA to bind in a non-specific way to the bacteria surface. For the extraction of GFP, column chromatography will be used, with aid of salt concentration that dictates protein-ligand interaction. Method Practical 1: Transformation of E.coli with Plasmid DNA Two closed microfuge were obtained and labeled “+pGLO” and the other “-pGLO” followed by placing them in a foam rack. The tubes were opened and a sterile pipette was used to transfer 250 ml of transformation solution (CaCl2) into each tube and then placed on ice for five minutes. A sterile loop was used to pick up a single colony from a plate with E.coli colonies. The +pGLO tube was picked and immersed into the transformation solution at the bottom of the tube. The loop was spin between index finger and thumb and then disposed off. The same procedure was repeated for the –pGLO tube while using a new sterile loop. Four agar plates were obtained and labeled: Label one LB/amp plate: +pGLO; Label two LB/amp/ara plate: +pGLO; Label three other LB/amp plate: -pGLO; Label four LB plate: -pGLO. Both the +pGLO and –pGLO tubes were transferred into a water bath set at 42 oC and the immersion was timed for exactly 50 seconds. The tubes were then replaced immediately back onto the ice. A suspension of LB broth mixed bacteria was spread evenly in each of the four plates. Practical 2: Purification of Recombinant GFP by Hydrophobic Interaction Chromatography 1 mL equilibration buffer [2 M (NH4)2SO4] was allowed to flow through in the chromatography column. 1.5 ml of E.coli culture was transferred to a microfuge. The bacteria was pelleted and the supernatant removed. 250 mL TE buffer was added and fixed with a blue tip. 50 mL lysozyme solution was added to the bacteria and the solution left to incubate at room temperature for 5 minutes. The tube was taken to the chemical safety cabinet for 1 minute followed by thawing the contents using hand warmth. The lysate of broken bacteria was spin 12,000 rpm for 5 minutes and transfered the supernatant to a microfuge. The less hydrophobic proteins were washed from column with low salt buffer. The GFP was eluted from the column with no salt buffer to separate it from the bacterial proteins. Results Appearance under UV light Tube Number Predicted Actual 1 Sample in binding buffer NO NO 2 Sample in wash buffer NO NO 3 Sample in elution buffer YES SLIGHT 4 Lysate pellet NO NO Discussion The successful achievement of DNA transfer, as shown by the resulting transformed bacteria, insinuates that a molecule as large as a plasmid can penetrate a cell membrane. Far from its weight, the plasmid carries negative charge of DNA which makes it impossible to penetrate the cell membrane freely. However, the combination of processes involved in the experiment was enough to settle this. First, the CaCI2 acting as the transformation solution produces positive charge ions that aid in neutralizing the negative charge. Another core step is incubation on ice, which acts in slowing the fluid cell membrane thereby making it easy for plasmid penetration. Applying heat shock also contributed to penetration possibility given its action in increasing the permeability of membranes. The required medium containing nutrients for bacterial growth and subsequent gene expression is also an important step to ensure successful transformation (Elumalai et al, 2014, 1410). In which case, the addition of LB broth proves useful because it contains the required amino acids and carbohydrates. In this case, column chromatography helped in purifying GFP from the many gene products found in E. coli. The hydrophobic interaction chromatography made purification possible given its action in concentrating GFP as the separation process continues (Noor, 2014, 1874). The process depends on salt concentration of buffer, which dictates protein-ligand interaction. In which case, the adding lysate pellet to the column resulted to no significant difference in UV-light. This occurred because it initiated only an interaction of salt ions with the less hydrophobic proteins and water. The addition of wash buffer, low salt buffer, also resulted to no detected green color because it made GFP to remain bind to the column while the rest of the proteins fall from the column. The addition of elution buffer produced positive results because it resulted to release of GFP from the column matrix. Elution makes use of no-salt buffer which dissipates protein-ligand interaction thereby leading to mobility of the protein (Bo, 2013, 44). Bibliography Stone, O, Biette, K, & Murphy, P 2014, Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein, Plos ONE, 9, 9, pp. 1-9, Academic Search Premier, EBSCOhost, viewed 26 November 2014. Noor, S, Tey, B, Tan, W, Ling, T, Ramanan, R, & Ooi, C 2014, Purification Of Recombinant Green Fluorescent Protein From Escherichia Coli Using Hydrophobic Interaction Chromatography, Journal Of Liquid Chromatography & Related Technologies, 37, 13, pp. 1873-1884, Academic Search Premier, EBSCOhost, viewed 26 November 2014. Bo, H, Wang, J, Chen, Q, Shen, H, Wu, F, Shao, H, & Huang, S 2013, Using a single hydrophobic-interaction chromatography to purify pharmaceutical-grade supercoiled plasmid DNA from other isoforms, Pharmaceutical Biology, 51, 1, pp. 42-48, Academic Search Premier, EBSCOhost, viewed 26 November 2014. Aune, T, & Aachmann, F 2010, Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed, Applied Microbiology & Biotechnology, 85, 5, pp. 1301-1313, Academic Search Premier, EBSCOhost, viewed 26 November 2014. Elumalai, S, Ramganesh, S, Prakasam, V, & Prabhakaran, M 2013, Construction of Plant Expression Vector of Synthetic Btcry1Ac Gene for Genetic Transformation, Advances In Bioresearch, 4, 2, pp. 44-52, Academic Search Premier, EBSCOhost, viewed 26 November 2014. Read More