FIG 10.

Reverse transcription to validate overexpression of asRNA in strain W83 harboring plasmid pTloop1 and ELISA showing reduced synthesis of K antigen when asRNA is overexpressed. (A) For comparison, total RNA was extracted from strain W83 harboring pT-COW (control) and W83 harboring plasmid pTloop1 grown to either the late exponential phase (OD₅₅₀ of 0.7) of growth or stationary phase (OD₅₅₀ of 1.1). Lane 1, 100-bp molecular weight marker; lane 2, control strain, stationary phase, asRNA; lane 3, control strain, late exponential phase, asRNA; lane 4, pTloop1 strain, stationary phase, asRNA; (5) pTloop1 strain, late exponential phase, asRNA; lanes 6 to 9, no-RT controls of samples in lanes 2 to 5; lane 11, 100-bp molecular weight marker; lane 12, control strain, stationary phase, 16S; lane 13, control strain, late exponential phase, 16S; lane 14, pTloop1 strain, stationary phase, 16S; lane 15, pTloop1 strain, late exponential phase, 16S; lane 16, genomic DNA, asRNA; lane 17, genomic DNA, 16S. Transcriptional analysis verified that strain W83 harboring plasmid pTloop1 overexpresses the asRNA encoded in the loop region during both late exponential and stationary phases of growth. (B) K-antigen capsule content of strain W83 with plasmid pT-COW or pTloop1 was quantitated by ELISA. The strain overexpressing the asRNA has reduced amounts of K-antigen capsule compared to the control strain. Error bars represent standard deviations of technical replicates.