We also tested other variables that might have affected the strength and utility of feeding. Interestingly, the current feeding vector does not have any transcriptional terminators, and thus we would expect transcripts to vary in size and also to contain sequences from the vector backbone. As a result, we tested a modified vector containing T7 terminators just outside the T7 promoters and discovered that inclusion of such terminators greatly diminished the effectiveness of RNAi (data not shown). Because it is also useful to concomitantly inhibit the functions of two genes, we tested the ability to feed two dsRNAs and learned that this greatly reduces the ability of each gene to independently produce a phenotype. Other methods could be tested for this purpose, however, including co-transforming two feeding vectors into the same bacteria or inserting two gene fragments into the same vector, either as single fragments or as inverted repeats.
