Chloramphenicol

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).

50 mg dried stems of WT and gux1/2 plants were used for each fermentation reaction. Stems were ball milled in 7 mL LB medium for 4 periods of 5 min at 20 Hz, with 5-min intervals between each ball milling cycle. The material was removed from ball milling vessel. To fully recover the biomass, the vessel was washed with further 2.5 mL LB. The stem suspension was sterilised by heat treatment at 85 °C for 10 min followed by cooling on ice. Each fermentation reaction was amended with 250 µL 1:10 Cellic^® CTec2 solution, prepared as described in saccharification section, and 250 µL of TOP10 E. coli inoculum bearing the BBa_K1122676 BioBrick (OD₆₀₀ of the inoculum was within 0.55–0.6 range). BBa_K1122676 encodes a Pyruvate decarboxylase and Alcohol dehydrogenase from Zymonomas mobilis which allow ethanol production in E. coli [25 (link)]. Biomass only reactions were supplemented with 250 µL of AmAc buffer and the bacterial inoculum. The plasmid was maintained by provision of 25 µg/mL Chloramphenicol (Duchefa Biochemie). The simultaneous saccharification and fermentation reactions were carried out for 96 h. at 37 °C and 200 rpm. Fermentation vessel was kept air-tight throughout the experiment. Ethanol levels were analysed using a commercial kit (Megazyme, catalogue code: K-ETOH).