Genetic transformation and competence in e. coli when exposed to the green fluorescent protein

Genetic transformation and competence in e. coli when exposed to the green fluorescent protein INTRODUCTION: The goal of this experiment was to successfully insert the plasmid pGLO, which carries genes for resistance to ampicilin and for green fluorescent protein (GFP), into competent E. coli cells thereby genetically transforming E. coli to have those specific traits. Green Fluorescent Protein comes from the jellyfish Aequorea Victoria and it emits green light when excited by blue light and when in the presence of the sugar arabinose. This protein has proven significant as a gene marker as well as other forth coming uses in biochemistry, cell and microbiology (Allison, Sattenstall, 2007). In a study done by Allison and Sattenstall (2007), it was found that introducing GFP into a cell causes changes in the cell physiology that might lead to antimicrobial susceptibility of the cell. This could be of concern because of its widespread use and Allison and Sattenstall urge caution when interpreting data from studies that used GFP (Allison, Sattenstall, 2007). According to Tsen et al., the E. c oli bacteria can naturally transform with inserted plasmids and integrate them without special treatments. As long as the DNA in the plasmids is Concatemeric linear, monomeric circular or supercoiled forms of plasmid, they can transform the E. coli, whereas linear monomer cannot transform it (Tsen, et al., 2002). The uses of green fluorescent protein in competent cells such as E. coli as mentioned before are very useful in gene markers and other studies in biochemistry, cell and microbiology, however, there are still being advances made for GFP. In a study done by Torrado, Iglesias and Mikhailov, techniques were improved in how well cells expressed the GFP gene based on the growing environments (Torrado, Iglesias, Mikhailov, 2008). In our experiment, we postulate that the E. coli will be competent for the pGLO plasmid carrying GFP and resistance to ampicilin. METHODS AND MATERIALS: In this experiment we will be using the plasmid pGLO which has genes for GFP and resistance to ampicilin. In order to force the plasmids into the E. coli cells, we will be using a heat shock treatment. This heat shock treatment causes the pores in the E. coli cell membrane to open, which allows the plasmid pGLO to enter the cell. We will test an E. coli centrifuge tube that has been exposed to pGLO and a centrifuge tube that has not been exposed to pGLO as our control. We labeled two centrifuge tubes with +pGLO and -pGLO to represent which tube carried the plasmid and which was our control, respectively. We first pipette 250 microliters of transformation solution into the tubes and added approximately 2 pen tip sized E. coli colonies. We then added the pGLO plasmids to the tube labeled +pGLO and rested both tubes on ice. The ice will make it so that the heat shock will have a greater affect on the E. coli, thereby increasing our chances of successful entrance of the plasmid. We then applied the heat shock treatment to the two centrifuge tubes by putting them in a 42ÂÂ °C water bath for 50 seconds. Afterward we put them back into the ice bath and prepared to put them into the four prepared agar plates. The four agar plates were split into two sets, two help +pGLO and two held our control -pGLO. The first plate contained just Luria Broth (LB) and 250 microliters of the -pGLO substance. The second control plate was LB with a mix of ampicilin (amp) and -pGLO E. coli solution which will ideally not grow any E. coli because E. coli by itself is not resistant to ampicilin. The third plate is used with +pGLO solution and is another LB/amp plate. The fourth and final plate is another +pGLO plate and contains LB and ampicilin but also arabinose, which is needed for the expression of the GFP. RESULTS: In this experiment, we used heat shock treatment in order to insert the pGLO plasmid into E. coli cells because the plasmid carries the genes that code for green fluorescent protein and ampicilin resistance. After a week of incubation in the refrigerator, we analyzed our four Petri dishes. All plates came out as predicted. Our first control plate (-pGLO E. coli cells) contained Luria Broth and ampicilin and it sustained 0% E. coli cell growth. The second control plate (-pGLO E. coli cells) contained only Luria Broth and there was 100% coverage of the agar plates. The lawn made by the E. coli cells was a whitish clear color in normal light and were not fluorescent green when exposed to UV light. In the two transformation plates, we received positive results matching our predictions. In the first transformation plate (+pGLO E. coli cells) there was a presence of Luria Broth and ampicilin. There were roughly 140 colonies of E. coli cells which was about 60% coverage of the Petri dish. Each of the colonies was an off whitish color under normal light but were not fluorescent green under UV light. The second transformation plate (+pGLO E. coli cells) contained a mixture of Luria Broth, ampicilin and arabinose. This plate had E. coli colony growth but there were only roughly 40 E. coli colonies, making about a 25% coverage of the plate. Again these colonies were whitish in color when exposed to normal light however, they did turn fluorescent green under the UV light. TABLE OF RESULTS: TYPE OF PLATE CONTENTS SKETCH OF PLATE OBSERVATIONS Transformation +pGLO/LB/amp/ara -Growth of Colonies (40 count; 25% coverage) -Whitish color in normal light -Fluorescent green under UV Transformation +pGLO/LB/amp -Growth in Colonies (140 count; 60% coverage) -Whitish color in normal light -No fluorescent green color under UV Control -pGLO/LB/amp -No E. coli growth, E. coli not transformed (0% coverage) Control -pGLO/LB -E. coli growth present (100% coverage) -Whitish color in normal light -No fluorescent color in UV light DISCUSSION: The hypothesis is the following: After heat shock treatment, the competent E. coli cells will receive the plasmid pGLO, and the E. coli cells will be transformed. We predicted that the E. coli cells would take in the plasmid and transform in our two transformation plates. In the -pGLO/LB control plate we predicted that significant growth would happen because there is no antibiotics and only an optimal growing environment. In the -pGLO/LB/amp control plate we predicted that there would be no growth of E. coli because ampicilin is present, an antibiotic that E. coli is not naturally resistant to. In the transformation plate +pGLO/LB/amp we predicted that there would be E. coli growth considering we hypothesized that the plasmid would be accepted by the E. coli cell, thereby giving it ampicilin resistance. In our last transformation plate +pGLO/LB/amp/ara we again expected growth of E. coli since we hypothesized the E. coli cell would be competent for the plasmid. We also expected that this would be the plate to glow fluorescent green since arabinose, the sugar that allows for the glowing, was present in the agar plate. In order for this experiment to show true results, we added the two control plates with different purposes. The first plate contained only Luria Broth, the ideal growing environment for E. coli. This plate was used to make sure that our E. coli cells were healthy and able to grow consistently. If they were unable to grow, that would mean that our cells were unhealthy or contaminated, which would in turn affect the results of our transformation plates. Our results for this plate were that we had healthy E. coli cells since they produced a full lawn. Our second plate was the one with both Luria Broth and ampicilin for the growing environment. We did not have any growth of E. coli on this plate, just as we predicted. This is good because the plasmid we were using to transform the E. coli cells have the gene that causes ampicilin resistance. If our E. coli had been contaminated or already transformed from its non-resistance state, we would see it in this control plate. If we had seen gro wth, we would know that our results for the transformation plates were faulty because our normal E. coli was already resistant. Next we examined our transformation plates. These plates were the ones that we exposed to the pGLO plasmid. Our first plate had Luria Broth and ampicilin, just like our control; however, since we treated this batch with the pGLO plasmid followed by heat shock treatment, we expected to see growth. Our results from this plate did show that the E. coli grew in the ampicilin agar plate, thereby showing how many of the E. coli cells received the plasmid and were able to be genetically transformed. However, under the UV light, the colonies did not glow fluorescent green because of the absence of arabinose. Our second transformation plate had Luria Broth, ampicilin and arabinose. Our results followed our predictions that we would see growth and have the colonies glow under the UV light. This is because the E. coli that took the plasmids were transformed so they showed their new resistance to ampicilin and they showed that when grown in an environment where arabinose is present, the green fl uorescent protein will be expressed. Our results did indeed support our hypothesis because the E. coli were transformed in our transformation plates because we saw that colonies were able to grow in an environment where ampicilin was present and also the green fluorescent protein was expressed in arabinose rich environments. There was an area of weakness in our experiment. The crucial part, the heat shock that opens the cellular membrane pores, could have gone a bit smoother. Our times were not exact due to many groups trying to do this part all at once. Secondly, there is room for error in the consistency of our experiment plates since each member of the group took turns at each stage of the process. In conclusion, the results of our experiment proved our hypothesis that the E. coli cells were competent for the pGLO plasmid. Our results were consistent with our predictions. We found that the E. coli cells can be transformed by the plasmid after our heat shock treatment. Our control plates can out controlled and our transformation plates produced colonies that expressed the GFP gene. SOURCES: Allison, D.G., Sattenstall, M.A. (2007). The Influence of green fluorescent protein incorporation on bacterial physiology: a note of caution. Journal of Applied Microbiology, 103(2), 318-324 Suh-Der Tsen, S., Suh-Sen Fang, S., Mei-Jye Chen, S., Jun-Yi Chien, S., Chih-Chun Lee, S., Han-Lin Tsen, D. (2002). Natural Plasmid Transformation in Escherichia coli. Journal of Biomedical Science, 9(3), 246-252. doi:10.1159/000059425. Torrado, M., Iglesias, R., Mikhailov, A.T. (2008). Detection of protein interactions based on gfp fragment complementation by fluorescence microscopy and spectrofluorometry. BioTechniques, 44(1), 70-74.