Design
Our designed constructs are linear dsDNAs, which can be separated into two parts, the upstream promoter and the downstream signaling component.
The following is our proof of concept design. The transcription is driven directly by T7 RNA polymerase from a strong T7 promoter, which then transcribing through an iSpinach aptamer surround by F30 scaffold sequences. The iSpinach transcript can bind with fluorophore, DFHBI. The fluorescence can be detected at 480/520 nm (1).
The purpose of this design is to proof that this linear construct can generate a complete transcript in our in vitro transcription system and emit detectable fluorescence.
Extending from the basic construct, we have incorporated metal sensing transcription factor binding site and riboswitch either upstream or downstream of the promoter to control the transcription of the RNA aptamer with the presence of metal ions.
ArsR is an arsenic (III) sensing transcription factor that belongs to one of the most diverse metal sensing repressor family, ArsR/SmtB. Upon binding to arsenic (III) metal ions, the DNA bound homodimer will dissociate from its DNA binding site that is overlapping with the -35 element recognized by RNA polymerase σ70 (2). ArsR was illustrated with the ability to sense 0.04-50 μM arsenic (III) in a whole cell biosensors through careful tuning of the TF /DNA template ratio (3).
Here, we took advantage of its DNA dissociation behaviour upon metal binding and combined it with a strong T7 promoter. The following is the design with ArsR binding site downstream of the T7 promoter, which were thought to have a great ability to prevent transcription leakage since the transcription factor is act like a road-block for the T7 RNA polymerase without the presence of metals.
Here we designed a construct consisting of a fluoride-responsive riboswitch, inspired by the research done by Walter Thavarajah, etc. (4). In nature, this riboswitch regulates the expression of the CrcB, a fluoride efflux pump in Bacillus cereus. In our design, it is positioned upstream of the reporter iSpinach aptamer.
This riboswitch is able to bind with fluoride ion co-transcriptionally, to form a secondary structure that allows the transcription machinery complex to pass. Which results in the transcription of downstream iSpinach. However, when fluoride is absent from the system, the terminator hairpin will be formed directly upstream of a polyuridine stretch instead, that will induce dissociation of the transcript from the template and the polymerase. Then the transcription is terminated. The 3 nucleotides inserted between the Anderson promoter and riboswitch sequence are helpful for the riboswitch to regulate the downstream gene expression.
