Tetracycline

To confirm the inability of escaped phages to infect E. coli, 10 μl of VHH-phage library was used to challenge 50 mL of E. coli XL-1 blue (OD₆₀₀ 0.45) for 30 min at 37°C. The challenged E. coli XL-1 blue were then plated on the LB (Sigma Aldrich) agar plate supplemented with Tetracycline: 50 μg/mL (Duchefa Biochemie BV), chloramphenicol: 50 μg/mL (Duchefa Biochemie BV) and Kanamycin: 50 μg/mL (Duchefa Biochemie BV).

Murashige and Skoog (MS) medium, spectinomycin, and sucrose were purchased from MB cell (CA, U.S.A). Tetracycline, cefotaxime and phosphinothricin were purchased from Duchefa (Haarlem, Netherlands). The pMJ103 vector was constructed by GreenGene BioTech (Yongin, Korea) and the plasmids (MYO51, MYO53) containing LTB and/or CTB were kindly donated from Dr. M.S. Yang (Chonbuk National University). T4 DNA ligase and restriction endonucleases were obtained from Roche (Basel, Switzerland). Gateway BP and LR recombinase systems were purchased from Invitrogen (Breda, Netherlands). A mini trans-blot cell and Mini-Protean III cell were purchased from Bio-Rad (Hercules, CA, USA). Reagents for SDS-polyacrylamide electrophoresis, such as acrylamide, bis-acrylamide, ammonium persulfate, TEMED, prestained molecular weight markers and Coomassie Brilliant Blue R-250 solution were obtained from Bio-Rad (Hercules, CA, USA). Anti-LTB polyclonal antibody was obtained from Abcam (Cambridge, MA, USA). G_M1-ganglioside and anti-rabbit IgG-conjugated alkaline phosphatase were purchased from Santa-Cruz Biotechnology (CA, USA). Thiamine-HCl, myo-inositol, anti-CTB polyclonal antibody, and other reagents such as salts and buffer components were of analytical grade and purchased from Sigma (St. Louis, MO, USA).