Ferrichrome Incubation as a Means to Reduce Phage Titer
Michael Muse, UC Berkeley, Gerald M. Rubin Lab Introduction During the production of the DGC set we found that 4 wells contained phage (160 pfu/µ). The following experiment demonstrates that the addition of ferrichrome (Sigma # F-8014) to the culture media at 1 µ can effectively reduce the titer of a phage contaminant. Ferrichrome does not destroy the phage, but rather it binds to the FhuA receptor of E. coli thereby preventing the binding of phages T1, T5, and Phi-80. We tested this through a single inoculation cycle consisting of 1 and 2 hour incubation periods and we were able to reduce the phage titer by approximately 50%. We included ferrichrome in the media for the source plates which were to serve as the progenitors of all of the copies of the DGC set. It is important to recognize the fact that the phage are still present in the culture media, and that they are still viable. The number of phage particles in a sample should remain constant throughout incubation. Multiple re-inoculations in growth medium containing ferrichrome should dilute the phage until very few remain. Please see references listed below. The Experiment We cultured a DH5-alpha clone overnight at 37° C and 250 rpm in LB medium with chloramphenicol (CAM) at a concentration of 25 µ/ml. The next day we spiked a new culture using the O/N growth of the DH5-alpha clone. We grew this culture at 37° C and 250 rpm until the OD600 was between 0.4 - 0.8 (making sure that it did not exceed 1.0). We put these cells on ice and later used them as lawn cells for the plating of our phage. We then inoculated 2 parallel 3 ml cultures using 10 µ of a phage contaminated clone. Both cultures contained CAM at 25 µ/ml, and were incubated at 37° C and 250 rpm. One of the two cultures also contained ferrichrome [Sigma # F-8014] at a concentration of 1µ We plated both cultures after one hour of incubation, and again after two hours incubation. We then incubated the plates overnight at 37° C. After 12-16 hours incubation we checked the titer of the phage. We found that the titer of the phage was approximately 50 percent lower for the cells cultured in the presence of ferrichrome. Bibliography
- Boulanger, P., Maire, M,. Bonhivers, M,. Dubois, S., Desmadril, M,. and Letellier, L. (1996). Purification and Structural and Functional Characterization of FhuA, a Transporter of Escherichia coli Outer Membrane. Biochemistry 35, 14216- 14224.
- Killmann, H., Videnov, G., Jung, G., Schwarz, H., and Braun, V. (1995). Identification of Receptor Binding Sites by Competitive Peptide Mapping: Phages T1, T5, and Phi-80 and Colicin M Bind to the Gating Loop of FhuA. J. Bacteriol. 177, 694-698.
- Mondigler, M., Holz, T., and Heller, K. (1996). Identification of the Receptor-Binding Regions of pb5 Proteins of Bacteriophages T5 and BF23. Virology 219, 19-28.