Inspiration:
Team Stanford-Brown 2006 & 2007

The Standard-Brown design make use of three relatively well characterized promoters, BAD/AraCp, lacp and tetp. By using the corresponding inducers, L-Arabinose, IPTG and aTc, respectively, transcriptions in the other two strands will be repressed, hence only a single fluorescence signal will be generated. However, the generation of outputs from their switch was inefficient. As a result, we started to look for potential problems and corresponding improvements that can be applied to the Brown design.

Potential Problem of the Brown's Design

After some literature review, we found out that the potential problem of their design maybe due to the use of BAD/AraCp. The major problem of BAD/AraCp is the dual function of AraC protein in which it can both activates and represses BADp. In the absence of arabinose, the dimeric AraC protein molecule will contact I₂ and O₂ half-sites on the DNA which generates a DNA loop that interferes with the access of RNA polymerase to the two promoters in the looping region, thus, repressing BADp. When arabinose is present, AraC will bind to I₁ and I₂ half-sites on the DNA, which allows the binding of RNA polymerase and thus will stimulates the transcription of BADp. The dual function of AraC protein complicates the behavior of BAD/AraCp which may pose potential threat to the stability of the Tristable switch.

In contrast, for TetR and LacI, both of the protein can only act as a repressor to their corresponding promoters. In the presence of their corresponding inducers, aTc and IPTG, respectively, it will reduce the affinity of the repressor binding to the operator sites of the promoter and thus activate transcription.

Our Design

Our design aims to achieve three main characteristics: stable and distinct signal output, and fast state-switching rate. In which stability is the major goal that we would like to achieve. As a results, the strength of the promoters need to be similar. The tristable switch was built in 3 modules, separated according to the promoter that drives its expression. For characterization of all smaller constructs, a medium-strength RBS (BBa_B0032) was used.

We have used fluorescence proteins, mRFP, mTagBFP and sfGFP as our reporters to indicate the signal outputs generated by the Tristable switch. As the result, overlapping in the excitation wavelength of the fluorescence proteins should be minimized.