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Determination of Fd‐tet Bacteriophage Infectivity of E. coli B91BK Under Conditions that Disrupt the Biotin‐Streptavidin Interaction
Author(s) -
Gunby Taylor Marie,
Soendergaard Mette
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.635.5
Subject(s) - streptavidin , infectivity , biotin , bacteriophage , escherichia coli , chemistry , serial dilution , biotinylation , formamide , chromatography , microbiology and biotechnology , biology , biochemistry , virology , virus , medicine , alternative medicine , organic chemistry , pathology , gene
Bacteriophage (phage) are commonly used in techniques such as phage display due to their ability to express large foreign proteins on various coat proteins, and to be easily propagated in E. coli . Validation of selected phage particles in binding assays often involves biotinylation of the virion to facilitate probing by streptavidin. Phage may be collected following validation assays and propagated for further analysis by breaking the biotin‐streptavidin interaction. However, little is known about the effects on E. coli infectivity of the harsh conditions (95% formamide, >90°C) typically utilized to disrupt the biotin‐streptavidin complex. The E. coli (K91BK) infectivity of wild type (WT) Fd‐tet phage was determined in Tris buffered saline (TBS; pH 7.5), and 10 mM ethylenediaminetetraacetic acid (EDTA), 95% formamide (EDTA‐formamide) at temperatures up to 100°C. In brief, 10 12 phage particles (virions) were incubated in TBS or EDTA‐formamide for 10 min at 25–100°C (5°C increments). The infectivity of E. coli was then determined by quantifying the number of transducing units (TU). To do so, various dilutions of phage were incubated with E. coli K91BK (OD 600 =0.2 of 1/10 dilution) in terrific broth (TB) and allowed to infect for 10 min at room temperature. Next, NZ amine, yeast extract (NZY) medium was added and then incubated at 37°C for 30 min to promote propagation. Finally, the culture was plated on NZY agar plates with 100 μg/mL kanamycin and 20 μg/mL tetracycline and grown overnight at 37°C. The resulting colonies were counted, and used to calculate TU/mL. Results showed that the TU/mL was lowered significantly (p<0.001) at temperatures above 75°C in TBS, indicating that the E. coli infectivity was decreased by temperatures above this range. Results also showed that the TU/mL was decreased in EDTA‐formamide, although this difference was not significant (p>0.05). This is mostly likely due to reduced TU/mL already observed at 25°C in EDTA‐formamide compared to TBS, thereby lowering the relative decrease. While a trend of lower TU/mL for EDTA‐formamide compared to TBS was observed, this difference was not significantly lower at high temperatures (>75°C; p>0.05), indicating that the former does not comparatively decrease E. coli infectivity. At temperatures known to disrupt the biotin‐streptavidin interaction (>90°C), both treatments reduced the infectivity compared to 25°C from approximately 10 10 to 10 9 TU/mL, corresponding to a factor of 10. While these results show that increasing temperature overall decreases the number of phage capable of transduction, a sufficiently high number of virions maintain the ability to infect E. coli . Thus, biotinylated phage may be released from streptavidin by disrupting the interaction using temperatures above 90°C and utilization of EDTA‐formamide. Such phage may subsequently be amplified by propagation in E. coli and utilized in further experiments. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .