theophylline riboswitch construct Fig Share.pdf

<b>Synthetic ReDirect Theophylline riboswitch cassette (pUC57-Apr-Theo)</b> For replacing native promotors with a theophylline riboswitch controlled (Rudolph et al., 2013) promoter (ErmE; Bibb et al., 1985) using the ReDirect technology described by Gust el al., (2003). See the Genbank file created by Ryan Seipke ([e-mail]) available here (pUC57-AprTheo.gbk). There is the potential to design the promotor replacement such that a hexahistidine tag can be added to your protein of interest, facilitating the detection of any resulting protein by western blotting against the hexa-His-tag. The primers can also be moved to remove this sequence from the construct when introducing to your strain of interest. The cassette is excised from pUC57 as an EcoRI/ HindIII fragment to use as a PCR template. Email Paul Hoskisson ([e-mail]) for the plasmid. The strategy is as a word document below with comments covering the features for simplicity and for checking at a glance, along with the sequence (See below). Manuscript Under Review – Thomas C. McLean, Paul A. Hoskissonand Ryan F. Seipke. Coordinate regulation of antimycin and candicidin biosynthesis. Submitted. <b> </b> <b>References</b> Bibb, M. J., Janssen, G. R. &amp; Ward, J. M. Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. <i>Gene</i> <b>38</b>, 215–226 (1985). Gust, B., Challis, G. L., Fowler, K., Kieser, T. &amp; Chater, K. F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. <i>Proceedings of the National Academy of Sciences</i> <b>100</b>, 1541–1546 (2003). Rudolph, M. M., Vockenhuber, M.-P. &amp; Suess, B. Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. <i>Microbiology</i>, <b>159</b>, 1416–1422 (2013).