theophylline riboswitch construct Fig Share.pdf
Synthetic ReDirect Theophylline riboswitch cassette (pUC57-Apr-Theo)
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 (R.Seipke@leeds.ac.uk) 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 (paul.hoskisson@strath.ac.uk) 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. Hoskisson and Ryan F. Seipke. Coordinate regulation of antimycin and candicidin biosynthesis. Submitted.
References
Bibb, M. J., Janssen, G. R. & Ward, J. M. Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. Gene 38, 215–226 (1985).
Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proceedings of the National Academy of Sciences 100, 1541–1546 (2003).
Rudolph, M. M., Vockenhuber, M.-P. & Suess, B. Synthetic riboswitches for the conditional control of gene expression in Streptomyces coelicolor. Microbiology, 159, 1416–1422 (2013).
