Difference between revisions of "Team:Hong Kong HKUST/Ideas and Mechanism"
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<b><em>Our Design</em></b> | <b><em>Our Design</em></b> | ||
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<p style="font-size:1.2em;">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. | <p style="font-size:1.2em;">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. | ||
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| + | <img src="https://static.igem.org/mediawiki/2016/7/79/T--Hong_Kong_HKUST--phlfp.png" alt="phlFp promoter" style="width: 334px; height:302px;"> | ||
| + | <a href="https://2016.igem.org/Team:Hong_Kong_HKUST/pPhlF" style="text-align: center; color: #72c9b6;">phlFp Profile</a></td></td> | ||
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| − | + | <img src="https://static.igem.org/mediawiki/2016/thumb/6/6d/T--Hong_Kong_HKUST--ptet.png/600px-T--Hong_Kong_HKUST--ptet.png" alt="Tetp promoter" style="width: 334px; height:302px;"> | |
| − | + | <a href="https://2016.igem.org/Team:Hong_Kong_HKUST/pTet" style="text-align: center; color: #72c9b6;">Tetp Profile</a></td> | |
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| − | < | + | <img src="https://static.igem.org/mediawiki/2016/thumb/1/18/T--Hong_Kong_HKUST--plac.png/600px-T--Hong_Kong_HKUST--plac.png" alt="Lacp promoter" style="width: 334px; height:302px;"> |
| + | <a href="https://2016.igem.org/Team:Hong_Kong_HKUST/pLac" style="text-align: center; color: #72c9b6;">Lacp Profile</a></td> | ||
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Revision as of 19:19, 19 October 2016
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 a 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 w, which generates a DNA loop, interfering the access of RNA Pol 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.
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.
1. 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.
2. 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.
