Figure 4.

Recombinant ELVd-Spinach RNA production in E. coli using the viroid-derived system. (a) Time-course analysis of the RNA that accumulated in E. coli cells that co-expressed ELVd-Spinach RNA and eggplant tRNA ligase. Aliquots were taken from the culture at different time points and RNA was extracted. RNAs were separated by denaturing PAGE and the gel was stained with ethidium bromide. Lanes 1 to 8, RNAs from the aliquots taken at 0, 2, 4, 6, 8, 10, 12 and 24 h post-induction of tRNA ligase expression. The positions of the circular and linear ELVd-Spinach RNAs are indicated on the right. E. coli cultures were grown at 37 °C in TB medium and induced with 0.1 mM IPTG at OD₆₀₀ 0.1 mM. Each lane contains the RNA that corresponds to 0.4 ml of culture. (b) Pictures of the sedimented E. coli cells that co-expressed tRNA ligase and an empty vector, ELVd or ELVd-Spinach, as indicated (left), and the corresponding RNA extracts (right). The lower pictures were taken under UV illumination using a GFP filter. DFHBI was added to either the E. coli culture before cell recovery (left) or the RNA extract (right).