Table 2.
Experiment | Index values
|
|||
---|---|---|---|---|
4–8 h | 5–8 h | 21 h | 24 h | |
Time course | ||||
No T7 promoter | 1.2 | 1.3 | ||
E. coli C5 32/−2 | 2.7 | 13.0 | ||
EGyr241/211 | 4.0 | 17.0 | ||
EGyr241/223 | 4.3 | 14.0 | ||
Synth C5 21/45 | 2.4 | 2.6 | ||
Dose effects | ||||
No T7 promoter | 1.6 | |||
EGyr241 | 6.9 | |||
EGyr241/223 | 10.0 | |||
EGyr241/223 C5 32/−2 | 26.0 | |||
No T7 promoter | 1.7 | |||
E. coli C5 32/−2 | 5.6 | |||
E. coli 32/32 | 7.9 | |||
Nucleotide level specificity | ||||
No T7 promoter | 1.3 | |||
EGyr241 | 7.4 | |||
EGyr241 var1 | 14.0 | |||
EGyr241 var2 | 6.3 | |||
EGyr241 var3 | 6.3 | |||
SGyr241 | 1.3 | |||
Species specificity | ||||
No T7 promoter | 1.4 | 1.1 | ||
EGyr241/211 | 2.0 | 23.0 | ||
EGyr241/223 | 2.1 | 7.1 | ||
SGyr241/EGyr211 | 1.7 | 0.9 | ||
SGyr241/EGyr223 | 1.6 | 1.2 |
Summary of multiple assays in vivo, listed as inhibition index values from quantitative plate cultures, as described in Table 1. Results represent two separate experiments done on different days. Time course: Panel from assays performed early (5–8 h) or late (24 h) after cultures were split and grown in parallel either with or without IPTG to induce EGS expression. Negative control transformants include no T7 promoter, and synthC5 21/45. Dose effects: Comparison between inhibition of growth of transformants containing vectors expressing one, two, or four EGSs (EGyr241, EGyr241/223, and EGyr241/223 C532/−2, respectively). Also comparison between transformants with vectors encoding two EGSs, either different (C5 32/−2) or identical (C5 32/32) in their EGS sequences. Nucleotide level specificity: Assessment of effects of various EGSs directed against the mRNA for gyrase A, designed to cleave position 241. Bacterial species specificity was addressed with EGyr241 and SGyr241, as explained in Fig. 2 and text. Tolerance of nucleotide mismatches was assessed with EGyr241var1, 2 and 3 constructs, as per Fig. 1. Species specificity: Extension of EGyr241 and SGyr241 studies to dual EGS vectors, expressing EGSs against either E. coli or S. typhimurium gyrase mRNA sequences.