Additional Library Screens to Isolate Optimized Riboswitches for Controlling Virulence Sample Clauses

Additional Library Screens to Isolate Optimized Riboswitches for Controlling Virulence. If the single clone construct fails to show significant upregulation of vir genes, a randomized library will be screened in front of virG and functional riboswitches will be selected based on virBp::lacZ activity. A similar strategy was employed to optimize the cheZ riboswitches discussed in Chapter 2. Screening a library in front of virG instead of lacZ would screen for virulence activation as opposed to riboswitch function. Plating the library without theophylline on bromo-chloro-indolyl-galactopyranoside (X-gal), a sub- strate that when hydrolyzed by β-galactoside produces a blue color, will screen for clones that show no virulence activity in the absence of ligand. The whitest colonies will be screened as described in section 2.4. Once identified these riboswitches can be cloned in front of lacZ and assayed individually in order to identify their specific properties. This screen can also be repeated in E. coli to optimize this system for that organism. List of Primers Used pISG38 5´-AAGATTATCTTCTGCAAGGAGGCAACAAGATGTGCGAACTCGAACTCG-3´ pISG39 5´-GCATCGTCTTGATGCCCTTGGCAGAAGATTATCTTCTGCAAGGAGGCA ACAAGATGTGC-3´ pISG40 5´-CGACTCACTATAGGTACCUCUUATACCAGCATCGTCTTGATGCCCTTGGC- 3´ ST412 5´-GTCGCCATCGATCGGGCCCTGAGGCCTGCAGCGGCCGCTACTAGTATT XXXXXXXXXXXXXXXXXXXXXXXXXXXXX-0´ XX000 5´-GGAATTCGCGGCCGCTTCTAGAGATACGACTCACTATAGGTACCGGTG ATACC-3´ ST423 5´-CTTGGCAGCACCCGCTGCNNAGGGGGTNNCAACAAGATGTGCGAACT CGATATTTTACAC -3´ ST424 5´-GTGTAAAATATCGAGTTCGCACATCTTGTTGNNACCCCCTNNGCAGCG XXXXXXXXXXXX-0´ XX000 5´-CCTTGGCAGCACCCGCTGCNNAGGGGGTNNAACAAGATGTGCGAACT XXXXXXXXXXXXX-0´ XX000 5´-GTGTAAAATATCGAGTTCGCACATCTTGTTNNACCCCCTNNGCAGCGG GTGCTGCCAAGG-3´ ST473 5´-GGCAGCACCAAGGGACAACAAGATGTGC-3´ ST474 5´-GCACATCTTGTTGTCCCTTGGTGCTGCC-3´ ST475 5’-CCTTGGCAGCACCNNNNNNCAACAAGATGTGCG-3´ ST476 5´-CGCACATCTTGTTGNNNNNNGGTGCTGCCAAGG-3´ JPG007 5´- XXXXXXXXXXXXXXXXXXXXXXXXX-0´ XXX000 5´-CGACGGGATCGATCCCCCC-3´ References
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