Proof of Concept: Our Testing Construct

To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function (Lynch et al, Chem Biol. 2007 Feb;14(2):173-84.), and Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (Topp et al, Appl. Environ. Microbiol. December 2010 vol. 76 no. 23 7881-7884). The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.

We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.



We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.