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 ([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. 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).