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INCREASING IRON BINDING CAPACITY OF E. COLI BACTERIA SABRİ ÖZKAN BESLER PROJECT SUPERVISOR: ZEHRA SAYERS TEACHING ASSISTANT: GÖKŞİN LİU Introduction Iron is a significant element for mostly all the organisms. It is used in several biological processes such as cell respiration, photosynthesis, DNA synthesis, gene regulation and tricarboxylic acid cycle, etc. Normally, bacteria can bind iron however, some bacteria have special proteins that bind the iron which increases the iron binding capacity. We took one of this genes which is responsible for synthesizing a protein called Ferric Binding Protein (FBP) from H. Influenzae and add this gene with a histidine residue at the end of the polypeptide chain to E. Coli bacteria with recombinant DNA method. Adding the FBP gene to the E. Coli makes the bacteria capable to synthesize the FBP. Figure 3: Purified Ferric Binding Protein Effects of Magnetic Field On E.coli with FBP and Without FBP Since iron is affected from the magnetic field, we assumed that if our bacteria can bind iron, they might be attracted by magnetic field. We prepared four cultures and separate them into two groups in which two of them was prepared with LB and the other two are prepared with LB and iron(II)chloride and one culture of each group was induced with IPTG, which is a chemical that stimulates the protein expression and starts the protein synthesis. After the induction step, we examined our samples under microscope and we applied magnetic field on our samples to see if any attraction happened. Figure 1: Ferric Binding Protein of H. Influenzae Results and Discussion • At the end of the magnetic field tests, we could not observe a distinct attraction to the magnet. We waited for one more day and examined the samples under microscope with a magnet again however, we could not observe a distinct movement change on our bacteria again. By the way, we made this procedure before this experiment and we observe a polarization on the bacteria that is why we repeat this procedure, thus we are planning to make this procedure again. • We checked our FBP with SDS PAGE. We expected to see 37 kDa lines on the gel since the molecular weight of the FBP is approximately 37000 Dalton. At the end of the procedure we saw the lines which means we successfully purified our Ferric Binding Protein. Protein Purification: After the FBP is synthesized from the bacteria, it can be purified with protein purification method. In this method, a liquid bacteria culture with FBP is collected and centrifuged. Afterwards the pellet of the bacteria is separated from the supernatant and the pellet is lysed. When the bacteria are lysed, they are centrifuged again and the supernatant is collected which contains the FBP. However, the supernatant does not only include FBP, it contains other components too. Nickel Affinity Column purification is a method for purifying our protein. Histidine has a high affinity to nickel beads, thus the FBP proteins which contain histidine residues can easily bind to nickel beads. After the proteins bind the beads, the FBP is separated from the beads by a special buffer called Elusion Buffer, which contains a high concentration of imidazole. Since we add high concentration of imidazole to the column, the proteins have to be separated from the imidazole and they are separated from imidazole with dialysis. At the end of the dialysis, we obtain purified Holo-FBP which has a pink color. After we obtain the purified FBP protein, we make SDS PAGE in which our protein samples are separated with electrical field by their molecular weights. References Krewulak, K. D., & Vogel, H. J. (2008). Structural biology of bacterial iron uptake. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1778(9), 1781-1804. doi:10.1016/j.bbamem.2007.07.026 Shouldice, S. R., Skene, R. J., Dougan, D. R., Snell, G., McRee, D. E., Schryvers, A. B., & Tari, L. W. (2004). Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens. Journal of Bacteriology, 186(12), 3903-3910. doi:10.1128/jb.186.12.3903-3910.2004 Skaar, E. P. (2010). The Battle for Iron between Bacterial Pathogens and Their Vertebrate Hosts. PLoS Pathogens, 6(8), e1000949. doi:10.1371/journal.ppat.1000949 Figure 2: E. Coli Bacteria with FBP synthesis capability under the microscope.
