Ramaldes, Alessandro April 2, 2018 DNA Transformation and Protein Purification 1. Introduction Bacterial genetic transformation was first introduced by Frederick Griffith in 1928 and since then it has revolutionized science and how humans interact with the world around them (Hensel, G, 2011). This genetic transformation consists of a genetic alteration of the cell by the incorporation of an exogenous genetic code from another organism which enters the cell through the cell membrane in a process known as Horizontal Gene Transfer (Margulis, 2009). Genetic transformation is broadly used in the industry to produce specific organisms with a desired characteristic and it is significantly important from an economic and health perspective. Crops producing larger yields in less soil, presenting pathogens a resistance and or resistance drastic environmental changes such as drought and high or low temperatures, bacteria producing proteins unknown for their natural metabolic process for pharmaceutical purposes are a few examples of where genetic transformation is being used in the industry and academia. The process of genetically modified an organism by transfer of genetic material between two or more non-related species is known as Transgenic process and can occur in different ways (Mchughen, 2007). By natural competence, when an organism in its normal physiological state take DNA material from its surroundings and such DNA is recombined into the organism own genome. By protoplasmic transformation, when enzymatic removal of the cell wall exposes the cytoplasmic membrane and allow the cell to take DNA in the presence of polyethylene glycol and by electroporation when the bacterial cell is mixed with a plasmid and submitted to a period of stress caused by a high-voltage charge (Edmisten, 2016). Bacteria is often the target for genetic transformation in academia because they are single celled organisms and have a fast reproduction period which makes possible to analyze if a specific trait passed down through generations. One of the most common plasmid used in genetic transformation for education purposes is the pGLO plasmid, which contains three genes that are Ramaldes, Alessandro April 2, 2018 joined together as Bla which codes for the beta-lactamase enzyme which will confers to the bacteria resistance to the beta-lactam family of antibiotics such as penicillin, araC which function as a promoter and regulates the expression of the Green Fluorescent Protein (GFP) also present in the pGLO plasmid. In this experiment a plasmid (pGLO) containing the gene which codes for Green Fluorescent Protein (GFP) from a bioluminescent Jelly fish (Aequorea Victoria) will be added to a broth mixture containing Escherichia coli and submitted to a series of transformation steps to analyze if the organism with transform and take the ampicillin resistance and fluorescent characteristic attributed to the pGLO plasmid and later on verify if this protein is possible to be purified for further studies. 2. Methods. (Maxwell, 2016) Part: I (transformation) Two microcentrifuge tubes received 250μL of 50mM CaClâ‚‚ each and were labeled + pGLO and – pGLO and set on ice. Two to four colonies of bacteria sample were inoculated in both tubes, however + pGLO tube also received 10μL of pGLO Plasmid (solution concentration of 0.8μg/μl) and tubes were incubated for 10 minutes on ice. Using a heating plate tubes were heat shocked for 50 seconds at 42°C and placed back on ice for two minutes incubation period. Afterwards 250μL of LB broth was added to each tube and incubated for 30 minutes at 37°C in a shaking incubator. Six agar plates were labeled: LB/AMP/AVAS +pGLO, LB/AMP/AVAS – pGLO, LB/AMP +pGLO, LB/AMP – pGLO, LB +pGLO, LB – pGLO. 100μL of the transformation substance and control were pipetted into their appropriated agar plates and evenly spread with the assistance of a sterile cell spreader and incubated at 37°C for 48 hours. Ramaldes, Alessandro April 2, 2018 Part: II (pGLO transformation efficiency calculation) LB/amp/Ara agar plate was placed under Ultraviolet light to verify the presence of fluorescence and afterwards counted the number of colonies in the plate. The total amount of DNA is calculated as DNA(μg) = concentration of DNA x Volume of DNA μl. Not all DNA is transformed so to determine the fraction of DNA the volume of spread LB/amp/ara plate is multiplied by the total volume in test tube, what result in the value to determine the amount of pGLO spread, that is the total amount of DNA used multiplied by fraction of DNA. The efficiency of the pGLO transformation is found by dividing he CFU by the amount of pGLO in μg. After calculation two to three of the fluorescent colonies were inoculated in 5ml of LB/amp/ara broth and incubated for 48 hours for next step. Part: III (Column Chromatography) Before starting the chromatography procedure, the broth was analyzed under UV light to verify if fluorescence was visible. Four microcentrifuge tubes were labeled CFL, FT, W and E and 1000 μl of broth was transfer to a blank tube and centrifuged at 12.000rpm for 2 minutes. After centrifuge the tube was submitted to UV light to verify that the GFP is concentrated in the cell pellet. The supernatant is removed without disturbing the pellets and 250 μl of TE buffer was added and finger vortexed. Again, tube is submitted under UV light t secure that GFP is evenly distributed. 10 μl of lysozyme solution is added to the suspension and vortexed for 30 seconds and set on ice for incubation for 10 minutes then placed on water bath at 37°C for about 2 minutes. After incubation, tube was centrifuged for 10 minutes at 12000 rpm and column was equilibrated by adding 1mL of equilibration buffer and slowly drained and another 1mL of equilibration buffer was added and drained till 1mL mark. The incubated tube was once more checked under UV light to verify if this time the supernatant should glob and not the pellet. Carefully transfer 250μL of supernatant to the tube labeled as CFL (Cell Free Lysate). 250μL of biding buffer is added to the CFL tube and mix. The microcentrifuge tube labeled as FT is placed under the column and slowly 500μL of the sample is added to the column allowing it to flow to the FT tube. After that Tube labeled W was placed under the column, which received 250μL of wash buffer. The same process Ramaldes, Alessandro April 2, 2018 was done for tube labeled E (Elution), however 750 μl of TE Buffer was added to the column. All tubes were examined under Ultra Violet light to detect where GFP is present. 3. Results LB LB +amp LB +amp + ara pGLO + Growth/No Glow Growth/ No Glow (*86) Growth /Glow (*57) pGLO - Growth/No Glow No Growth No Growth Table 1: indication of the reactions and results from all six agar dishes. Note that only the plate containing pGLO +, LB, amp and ara presented growth with fluorescence. sections containing symbol (*) shows the number of bacterial colonies found in their respective agar dishes. Figure 1: Colonies of E. coli containing in LB, amp and araC displaying a fluorescence when exposed to the ultraviolet light Figure 2: Sample of purified Green Fluorescent protein under ultraviolet light displaying a week fluorescence (Table 2) Transformation Efficiency Calculation: TE= (CFU)/[pGLO] TE= (57 colonies /0.8 μg/ml) = 71.3 CFU/μg Ramaldes, Alessandro April 2, 2018 4. Discussion Escherichia coli developed normally in both dishes containing pGLO positive and negative in Luria-Bertani broth (LB). The growth is explained because there was no agent on the media and broth that would prevent the organism to naturally grow and thrive. Transformation was observed when media was analyzed under ultraviolet light, however, this result was expected since E. coli araC and amp was not present in the process. Analyzing the agar dish labeled LB/amp for pGLO positive is possible to see that Escherichia coli presented growth indicating that the organism transformed and became resistant to ampicillin antibiotic present in the Luria-Bertani (LB) broth due to the presence of beta-lactamase protein which the organism must have acquired through transformation. The Escherichia Coli studied in this experiment did not contain strains of natural resistance to ampicillin, so it should present a non-growth behavior, since antibiotics like ampicillin acts by preventing the bacteria to constructing a cell, what is confirmed when contrasting the pGLO negative dish with LB/amp wish did not develop growth because as a control it did not received the plasmid containing beta-lactamase protein code (table 1). Even though the dish containing pGLO + with LB/amp displayer growth and antibiotic resistance to ampicillin, it did not display the fluorescence indicating transformation for GFP. It can be explained by the fact that the organism did not receive the genetic code necessary to display or promote the desired phenotypic trait. Agar dish containing pGLO positive and LB/araC/amp did present growth of Escherichia coli and displayed fluorescence, indicating that this sample test went under the transformation process to become resistant to ampicillin and to produce the GFP (Figure 1). In this case the Green Fluorescent Protein was active due to the presence or araC which encodes the regulatory protein that binds to the pBAD promoter, an upstream GFP gene, that when attached to the araC-arabinose switch the GFP gene on conferring its fluorescent aspect (Schleif, 2010). The Transformation efficiency of this sample was found to be 71.3 CFU/μg , what shows a considerable amount of transformation of cell per microgram of DNA. Ramaldes, Alessandro April 2, 2018 The process of purification of GFP did not present a satisfactory result since during the process column chromatography the supernatant sample displayed a very low index of fluorescence, implying that the GFP was present but not in a high concentration (figure 2). This result was due to the probable non-addition and subsequent substitution of one buffer, very likely to be the Elution Buffer (EB) since it acts as a major solvent in affinity chromatography and it washes away unbound protein in a greater concentration and releases the desired protein from the ligand (Banks, 2012). By this fact not enough protein was extract from the pellets into the supernatant conferring a questionable or wrong result. To confirm that purification of GFP was possible with the methodology applied in this study, a sample containing GFP under the proper procedure was obtained from another group (group 4). The group’s sample presenter a brighter fluorescence indicating a higher concentration of purified GFP in the supernatant. 5. Conclusion Escherichia coli went under the transformation process performed in this experiment which conferred to the organism the capability of ampicillin resistance and the capability to produce GFP which was observed under the ultraviolet light. The purification of the protein, even though was not successful for the group to extract enough of the GFP from the pellet to the supernatant, was proofed to be possible when using a sample from another group which followed the same procedure, materials and environmental settings. Further studies regarding this experiment should analyze the limits of genetic transformation in relationship the capacity of Escherichia coli to uptake genes for different traits without fully changing the main genetic framework of the bacteria. Ramaldes, Alessandro April 2, 2018 6. References. Banks, C. A., Kong, S. E., & Washburn, M. P. (2012). Affinity purification of protein complexes for analysis by multidimensional protein identification technology. Protein Expression and Purification, 86(2), 105-119. doi:10.1016/j.pep.2012.09.007 Floyd, J, and Maxwell, R. (2016 Lab 08): GFP Transformation and Protein Purification. Georgia State Univerity – LabArchieves. Retrieved on March 08, 2018 from https://goo.gl/VpPYP6 Hensel, G. (2011). Genetic Transformation of Triticeae Cereals for Molecular Farming. Genetic Transformation. doi:10.5772/22430 Edmisten, K. (2016, July/August). What Is the Difference Between Genetically Modified Organisms and Genetically Engineered Organisms. Retrieved April 07, 2018, from https://agbiotech.ces.ncsu.edu/q1-what-is-the-difference-between-genetically-modifiedorganisms-and-genetically-engineered-organisms-we-seem-to-use-the-terms-interchangeably/ Margulis, L. (2009). Genome Acquisition in Horizontal Gene Transfer: Symbiogenesis and Macromolecular Sequence Analysis. Horizontal Gene Transfer Methods in Molecular Biology, 181-191. doi:10.1007/978-1-60327-853-9_10 Mchughen, A., & Smyth, S. (2007). US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnology Journal, 0(0). doi:10.1111/j.1467-7652.2007.00300.x Schleif, Robert. “AraC Protein, Regulation of the l-Arabinose Operon InEscherichia Coli, and the Light Switch Mechanism of AraC Action.” FEMS Microbiology Reviews, vol. 34, no. 5, 2010, pp. 779–796., doi:10.1111/j.1574-6976.2010.00226.x.
