Evaluation of biotin binding to bacteriophage for E.coli bacteria trapping in nanotechnology study

In first, E.coli bacteria and bacteriophage culture were done. Briefly, in ۱۰۰۰ mLi deionized water, ۲۰۰ mgr Nutrient broth was added. Then it was boiled and sterilized in Autoclave for ۱۵ min at ۱۲۱ ۰C. In next step, ۲ mLi E coli were added to mix in sterile in environment. So it was shaking at ۳۷ ۰C in ۱۵۰ rpm and stored at -۸۰ ۰C. For culture of E. coli bacteria (OD=۰.۵, λ=۶۰۰ nm), ۲۰۰ μLi bacteriophage was added and shake at ۳۷ ۰C in ۱۵۰ rpm for overnight.For isolation of bacteria carcasses, it was spin for ۱۰ min at ۵۰۰۰ rpm. Then, LB Broth, NaCl and PEG was added to supernatant and stored at ۴ ۰C for ۲ h and after dewatering of supernatant, it was stored at ۴ ۰C in PBS. Biotinylation of bacteriophage is the first step to immobilization. In this experiment, NHS-Biotin solution was added to the total volume of the bacteriophage ۱۰%, and stirred gently, and incubated at room temperature for ۴ hours. In this step, NHS-Biotin reacts with the phage surface protein through primary amines. Ideally for nanotechnology using, the head protein of phage will be biotinylated and the phage tail will be free to attachment of host bacterial cells. So, the biotin concentration is important to optimize binding of phage on electrode surface and also retaining of maximum phage activity. The optimum biotin concentration attachment on phage for maximum activity was done by cell culture. When the biotin concentration was low (<۲.۵ mg/ml), phage activity was not affected by the biotin used. The phage activity decreased ۱.۶ log cycles as the biotinconcentration increased to ۳۰ mg/ml. Therefore, ۲.۵ mg/ml biotin was chosen to biotinylated the bacteriophage.

کلیدواژه ها:

biotin ، bacteriophage ، E coli bacteria