Team:Hong Kong HKUST/Ideas and Mechanism
Inspiration:
Team Stanford-Brown 2006 & 2007
The Standard-Brown design made 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 one single fluorescence signal will be generated. However, the fluorescence output 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 reviewing some literature, we found out that the potential problem of their design might be due to the use of BAD/AraCp. The major problem of BAD/AraCp is the dual function of AraC protein, which can both activate and repress 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, interfering the access of RNA polymerase to the 2 promoters
, repressing BADp. When arabinose is present, AraC binds to I1 and I2 half-sites on the DNA, which allows the binding of RNA polymerase; thus, will stimulate the transcription of BADp. The dual function of AraC protein complicates the behavior of BAD/AraCp which pose potential threat to the stability of the Tristable Switch.
In contrast, both TetR and LacI, can only act as repressors to their corresponding promoters. In the presence of their corresponding inducers, aTc and IPTG, respectively, the affinity of the repressor binding to the operator sites of the promoter will be reduced 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, strengths 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.
1. phlFp
The major difference between the two designs are that we substituted BAD/AraCp with phlFp. As PhlF is a member of the TetR family repressors, therefore, the repressor behave similar as TetR in which the binding of the inducer DAPG with the repressor will lower the it’s affinity to bind to the operator sites of the promoter, in turn activates promoter transcription. Second, according to Brynne et al. (2014), the response functions of phlFp is comparable to tetp, as a result, it is possible to fine tune the strength difference of the promoters using different strength of RBS.
2. tetp
The tetp module consists of a Tet repressible promoter upstream of the CDSs of phlF and lacI, which are then followed by the reporter gene, mtagbfp. When tetp is actived, the two repressor proteins, PhlF and LacI will be expressed along with the reporter gene, mtagbfp. This results in the repression of the two other promoters in our system, phlfp and lacp.
Creating a reproducible Tristable Switch with distinct signals is another goal we would like to achieve this year. As the three states are interconnected with each other, high level of stochastic noise could possibly lead to indistinct signals and thus disrupt the stability of our genetic circuit. As a result, the tetp we used is an improved version with minimized background expression. With this, we hope to minimize the number of variants that may affect the stability of our genetic circuit so as to achieve the goal mentioned previously.
3. Reporters
We have used three 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.
REFERENCES
- Brynne C Stanton, Alec AK Nielsen, Alvin Tamsir, Kevin Clancy, Todd Peterson & Christopher AVoigt. (2014). Genomic mining of prokaryotic repressors for orthogonal logic gates. Nature Chemical Biology, Vol 10.
- Oksana M. Subach , Paula J. Cranfill , Michael W. Davidson & Vladislav V. Verkhusha. (2011). An Enhanced Monomeric Blue Fluorescent Protein with the High Chemical Stability of the Chromophore. PLoS ONE, Vol 6, Issue 12.
