Part:BBa_K2448025
Psicose Biosensor pPsiA-PsiR from Agrobacterium tumefaciens
This part is a Psicose Biosensors based on the PsiR transcription factor from Agrobacterium tumefaciens (BBa_K2448006) and its associated promoter pPsiA (BBa_K2448010).
Usage and Biology
Biosensors rely on a basic theoretical principle: a certain concentration of a molecule of interest induces the proportional production of an easily detectable output, like fluorescence. Transcription-factor based biosensors allow quick and cheap detection or quantification of various chemical compounds.
Psicose biosensors were the lynchpin of our iGEM project. In order to improve the enzymatic production of psicose, we needed an efficient method to screen large banks of mutants. To address this problem, we designed a collection of specific biosensors able to detect psicose concentration in vivo (BBa_K2448025, BBa_K2448026, BBa_K2448027, BBa_K2448028, BBa_K2448029, BBa_K2448030 an BBa_K2448031). This allowed us to correlate the fluorescence with the mutant enzyme activity. Combining this biosensor with a plate reader, we were able to screen a library of mutants and identify the cells carrying an improved version of our enzyme, the D-Psicose 3-epimerase (Dpe) from Clostridium cellulolyticum (BBa_K2448021. For more details see BBa_K2448035.
Features
This biosensor was built using the Universal Biosensing Chassis (BBa_K2448023, BBa_K2448024) which is a composite part that provides an answer to the lack of rapid and reliable building methods for transcription-factor based biosensors.
This part is a Psicose Biosensors based on the PsiR transcription factor from Agrobacterium tumefaciens (BBa_K2448006) and its associated promoter pPsiA (BBa_K2448010).
PsiR from Agrobacterium tumefaciens (BBa_K2448006) is a predicted LacI family transcription factor with high affinity for D-Psicose. This implies that PsiR is potentially capable of binding a consensus sequence in the promoter region and preventing transcription of the regulated promoters in the absence of D-Psicose, in a similar manner to the way LacI does in the absence of allolactose (or the synthetic IPTG).
In this biosensor, we used this Helix-Turn-Helix transcription factor together with the Agrobacterium tumefaciens pPsiA promoter (BBa_K2448010) which is the promoter region (0.4 kb upstream) of the PsiA gene of Agrobacterium tumefaciens str. C58 (Atu4744). pPsiA is predicted to be repressed by the PsiR transcription factor (BBa_K2448006)which is inhibited in the presence of D-Psicose. This promoter regulates the expression of mCherry in our biosensor.
The results presented hereafter show that this duo PsiR - pPsiA behaved as predicted under and without induction.
Principle
When pTacI is induced by IPTG, it drives the transcription of the PsiR gene coding for the PsiR protein which is predicted to be a transcription factor able to bind D-Psicose. If D-Psicose is present in the cell, the transcription factor will bind preferentially to it and thus be inactivated. The repression of the related promoter pPsi will be released, enabling the transcription of a fluorescent protein, mCherry. If D-Psicose isn’t present in the cell, PsiR will bind to pPsi, preventing any transcription of mCherry.
Characterization
The detailed protocol is presented in the Experience page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1337
Illegal XhoI site found at 62 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 104
Illegal AgeI site found at 791
Illegal AgeI site found at 2351
Illegal AgeI site found at 2463 - 1000COMPATIBLE WITH RFC[1000]
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